Note: Descriptions are shown in the official language in which they were submitted.
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PREDICTIVE BIOMARKERS FOR BREAST CANCER
REFERENCE TO SEQUENCE LISTING
The present application is being filed along with a Sequence Listing in
electronic format. The
Sequence Listing is provided as a file entitled 20151006PCTSEQLST.txt, created
on October 15,
2012, which is 39.6 KB (40,639 bytes) in size. The information in the
electronic format of the
sequence listing is incorporated herein by reference in its entirety.
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional Patent Application No.
61/547,838 filed
October 17, 2011 and U.S. Provisional Patent Application No.: 61/605,798 filed
March 2, 2012, each
of which are incorporated by reference in its entirety.
FIELD OF THE INVENTION
The invention relates to compositions, methods, assays and kits for detecting,
screening,
diagnosing or determining the progression of, regression of and/or survival
from a proliferative
disease or condition, in particular breast cancer.
BACKGROUND OF THE INVENTION
Breast cancer is the most common form of cancer in women and is second only to
lung cancer as a
cause of death. It is estimated that, based on current incidence rates, an
American women has a one in eight
chance of developing breast cancer at some time during her life. According to
the American Cancer Society, in
2010, an estimated 207,090 new cases of invasive breast cancer were expected
to be diagnosed in women in
the U.S., along with 54,010 new cases of non-invasive (in situ) breast cancer.
As would be expected for such a major disease, the first efforts to apply
emerging molecular and
immunohistochemistry techniques in the 1980s to human cancers focused on
breast cancer. Initial work
considered the amplification of dormant oncogenes as prognostic markers and
subsequently featured
assessment of tumor suppressor genes. This was accompanied by a great interest
in invasion and metastasis
markers initially evaluated by immunohistochemistry and subsequently studied
by molecular biologic
techniques.
The morphologic features of primary breast cancer specimens and the pathologic
stages that are
determined from local and more extensive specimen resections have been the
fundamental predictors of
prognosis in the disease for more than a century. The morphologic prognosis
parameters or features remain the
cornerstone of predicting disease outcome. Current general consensus holds
that axillary lymph node
metastasis is the most significant morphologic prognosis parameter or feature,
followed by tumor type, tumor
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grade, and tumor size. Additional important morphologic parameters generally
considered to be predictive are
the extent of an intraductal component in patients with mixed intraductal and
infiltrating ductal carcinoma,
proven intralymphatic and intravascular invasion, and high mitotic index.
Ninety percent of breast cancers are
ductal in origin including infiltrative and mixed infiltrative and in-situ
cases. The impact of tumor type is not
significant for the vast majority of patients with the disease.
Despite the predictive value of morphologic prognostic evaluation,
approximately 25% - 30% of
patients with lymph node negative breast cancer will relapse and die from the
disease and approximately 25%
of patients with lymph node positive tumors will not relapse and die of the
disease.
Fatty acid synthase (FAS, FASN), a 270 kDa protein is found in tumor cells
from breast carcinomas
of patients whose prognosis is very poor. Although the biochemical and
metabolic basis for FAS expression in
tumor cells in not well understood, it appears that FAS expression confers a
selective advantage compared to
normal cells.
It is thought that FAS regulates progression of the cell division cycle from
the G1 into S phase by
binding to and thus inhibiting the cyclin E/Cdk2 complex. It has been recently
reported that reduced expression
of FAS correlates with poor survival in cohorts of breast carcinoma as well as
colon carcinoma patients.
Immunohistochemical analysis of FAS expression in primary breast cancers has
shown that low
nuclear expression of FAS protein is a significant predictor of very poor
disease-free survival. Results of a
study using tumors from a selection of younger women, which were selected on
the basis of a higher cancer
death rate, showed that decreased levels of FAS were a significant predictor
of poor overall survival by
multivariate regression analysis.
The recent advances in breast cancer detection have made it possible to detect
very small invasive
breast carcinomas, which has resulted in an increased number of diagnosed
cases of in-situ carcinoma. While
there are a number of prognostic markers already in use for breast cancer to
date, the need for a powerful
prognostic marker such as describe herein is apparent.
SUMMARY OF THE INVENTION
The present invention provides methods, assays and kits for the prediction of
clinical outcomes for patients
with early stage (node negative) breast cancer. These include assays which
involve immunohistochemical techniques
and may involve the use of FAS antibodies which may be labeled with a
detectable label.
In one embodiment is provided a method of predicting a clinical outcome of a
patient diagnosed
with breast cancer, the method comprising obtaining a tissue sample from the
patient by excision,
aspiration or biopsy, assaying the sample by one or more colorimetric methods
to determine the
nuclear stain intensity and stain positivity of fatty acid synthase, and
classifying as a good clinical
outcome any assay results where stain intensity and stain positivity are
individually at least 2.00.
Stain intensities and stain positivities may vary within certain ranges. These
ranges for stain
intensity include from about 2.45 to about 4. In this range the stain
positivity may be from about 3.69
to about 4. Further ranges for stain intensity include from about 2.69 to
about 4. In this range the stain
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positivity may be from about 3.23 to about 4. Further ranges for stain
intensity include from about
3.10 to about 4. In this range the stain positivity may be from about 3.00 to
about 4. Further ranges for
stain intensity include from about 3.23 to about 4. In this range the stain
positivity may be from about
2.55 to about 4. Further ranges for stain intensity include from about 3.69 to
about 4. In this range the
stain positivity may be from about 2.45 to about 4.
In one embodiment the sample from the patient is selected from the group
consisting of
epithelial cells or tissue, ductal components, lymph fluid and inflammatory
cells or combinations of
these. Patients may have been previously diagnosed with cancer which is lymph
node negative or
positive.
Assays of the present invention include immunoassays. These may include any
immunoassay
including but not limited to ELISAs, IHC or other colorimetric assay.
The antibodies used may be polyclonal or monoclonal antibodies and may contain
a
detectable label.
In one embodiment, a method for predicting the of likelihood of a metastatic
event occurring
within five years in a lymph node negative patient diagnosed with breast
cancer independent of age,
Her2/neu status or estrogen receptor status is provided comprising obtaining a
breast tissue sample
from the patient by excision, aspiration or biopsy, assaying the sample to
determine the nuclear stain
positivity boundary pair scores of fatty acid synthase, classifying the
results as likely to predict the
occurrence of a metastatic event in the patient when the boundary pair scores
are individually less
than 2.00.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a method for the stratification of patients
with breast cancer. The
invention provides a superior assay, involving the measurement and/or scoring
of FAS expression, for the
prediction of outcomes for patients with early stage (node negative; T1NOMO,
where (Ti) clinical
stage I; (NO) Node Negative; and (MO) is No Mets) breast carcinoma.
The methods provided here represent an improvement over mere morphologic
parameter or feature
assessments and those of mammography. Mammographic sensitivity ranges from 83%
to 95%, and specificity
ranges from 93% to 99%. Furthermore, sensitivity and specificity are lower in
women who are younger than
50. Thus, 5% to 10% of all screening mammograms are reported as abnormal, and
¨ more importantly- about
90% of women with abnormal mammograms do not have breast cancer. Appropriate
and timely follow-up of
abnormal mammograms is crucial for relieving patients' anxiety and for
assuring prompt intervention if
malignancy is present. Optimal strategies for managing patients with abnormal
mammograms should allow
clinicians to rapidly identify those patients who have breast cancer, and,
with the same speed and accuracy, to
identify (and reassure) those who do not. These strategies may now include the
methods of the present
invention.
Definitions
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Unless otherwise defined, all technical and scientific terms used herein have
the same meaning as
commonly understood by one of ordinary skill in the art to which this
invention belongs. Although methods
and materials similar or equivalent to those described herein can be used in
the practice or testing of methods
featured in the invention, suitable methods and materials are described below.
For convenience, the meaning of certain terms and phrases employed in the
specification, examples,
and appended claims are provided below. The definitions are not meant to be
limiting in nature and serve to
provide a clearer understanding of certain aspects of the present invention.
The term "genome" is intended to include the entire DNA complement of an
organism, including the
nuclear DNA component, chromosomal or extrachromosomal DNA, as well as the
cytoplasmic domain (e.g.,
The term "gene" refers to a nucleic acid sequence that comprises control and
most often coding
sequences necessary for producing a polypeptide or precursor. Genes, however,
may not be translated and
instead code for regulatory or structural RNA molecules.
A gene may be derived in whole or in part from any source known to the art,
including a plant, a
successful transcription and in most instances translation to produce a
protein or peptide. For clarity, when
reference is made to measurement of "gene expression", this should be
understood to mean that measurements
may be of the nucleic acid product of transcription, e.g., RNA or mRNA or of
the amino acid product of
The phrase "single-gene marker" or "single gene marker" refers to a single
gene (including all variants
of the gene) expressed by a particular cell or tissue type wherein presence of
the gene or transcriptional products
thereof, taken individually the differential expression of such, is
indicative/predictive of a certain condition.
30 The term "nucleic acid" as used herein, refers to a molecule comprised
of one or more nucleotides, i.e.,
ribonucleotides, deoxyribonucleotides, or both. The term includes monomers and
polymers of ribonucleotides
and deoxyribonucleotides, with the ribonucleotides and/or deoxyribonucleotides
being bound together, in the
case of the polymers, via 5' to 3' linkages. The ribonucleotide and
deoxyribonucleotide polymers may be single
or double-stranded. However, linkages may include any of the linkages known in
the art including, for example,
produced analogs that are capable of forming base-pair relationships with
naturally occurring base pairs.
Examples of non-naturally occurring bases that are capable of forming base-
pairing relationships include, but
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are not limited to, aza and deaza pyrimidine analogs, aza and deaza purine
analogs, and other heterocyclic base
analogs, wherein one or more of the carbon and nitrogen atoms of the
pyrimidine rings have been substituted by
heteroatoms, e.g., oxygen, sulfur, selenium, phosphorus, and the like.
The term "complementary" as it relates to nucleic acids refers to
hybridization or base pairing between
nucleotides or nucleic acids, such as, for example, between the two strands of
a double-stranded DNA molecule
or between an oligonucleotide probe and a target are complementary.
As used herein, an "expression product" is a biomolecule, such as a protein or
mRNA, which is
produced when a gene in an organism is expressed. An expression product may
comprise post-translational
modifications. The polypeptide of a gene may be encoded by a full length
coding sequence or by any portion of
the coding sequence.
The terms "amino acid" and "amino acids" refer to all naturally occurring L-
alpha-amino acids. The
amino acids are identified by either the one-letter or three-letter
designations as follows: aspartic acid (Asp:D),
isoleucine (Ile:I), threonine (MET), leucine (Leu:L), serine (Ser:S), tyrosine
(Tyr:Y), glutamic acid (Glu:E),
phenylalanine (Phe:F), proline (Pro:P), histidine (His:H), glycine (Gly:G),
lysine (Lys:K), alanine (Ala:A),
arginine (Arg:R), cysteine (Cys:C), tryptophan (Tip:W), valine (Val:V),
glutamine (Gln:Q) methionine
(Met:M), asparagines (Asn:N), where the amino acid is listed first followed
parenthetically by the three and one
letter codes, respectively.
The term "amino acid sequence variant" refers to molecules with some
differences in their amino acid
sequences as compared to a native sequence. The amino acid sequence variants
may possess substitutions,
deletions, and/or insertions at certain positions within the amino acid
sequence. Ordinarily, variants will possess
at least about 70% homology to a native sequence, and preferably, they will be
at least about 80%, more
preferably at least about 90% homologous to a native sequence.
"Homology" as it applies to amino acid sequences is defined as the percentage
of residues in the
candidate amino acid sequence that are identical with the residues in the
amino acid sequence of a second
sequence after aligning the sequences and introducing gaps, if necessary, to
achieve the maximum percent
homology. Methods and computer programs for the alignment are well known in
the art. It is understood that
homology depends on a calculation of percent identity but may differ in value
due to gaps and penalties
introduced in the calculation.
By "homologs" as it applies to amino acid sequences is meant the corresponding
sequence of other
species having substantial identity to a second sequence of a second species.
"Analogs" is meant to include polypeptide variants which differ by one or more
amino acid alterations,
e.g., substitutions, additions or deletions of amino acid residues that still
maintain the properties of the parent
polypeptide.
The term "derivative" is used synonymously with the term "variant" and refers
to a molecule that has
been modified or changed in any way relative to a reference molecule or
starting molecule.
The present invention contemplates several types of compositions, such as
antibodies, which are amino
acid based including variants and derivatives. These include substitutional,
insertional, deletion and covalent
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variants and derivatives. As such, included within the scope of this invention
are polypeptide based molecules
containing substitutions, insertions and/or additions, deletions and
covalently modifications. For example,
sequence tags or amino acids, such as one or more lysines, can be added to the
polypeptide sequences of the
invention (e.g., at the N-terminal or C-terminal ends). Sequence tags can be
used for polypeptide purification or
localization. Lysines can be used to increase solubility or to allow for
biotinylation. Alternatively, amino acid
residues located at the carboxy and amino terminal regions of the amino acid
sequence of a peptide or protein
may optionally be deleted providing for truncated sequences. Certain amino
acids (e.g., C-terminal or N-
terminal residues) may alternatively be deleted depending on the use of the
sequence, as for example, expression
of the sequence as part of a larger sequence which is soluble, or linked to a
solid support.
"Substitutional variants" when referring to proteins are those that have at
least one amino acid residue
in a native or starting sequence removed and a different amino acid inserted
in its place at the same position. The
substitutions may be single, where only one amino acid in the molecule has
been substituted, or they may be
multiple, where two or more amino acids have been substituted in the same
molecule.
As used herein the term "conservative amino acid substitution" refers to the
substitution of an amino
acid that is normally present in the sequence with a different amino acid of
similar size, charge, or polarity.
Examples of conservative substitutions include the substitution of a non-polar
(hydrophobic) residue such as
isoleucine, valine and leucine for another non-polar residue. Likewise,
examples of conservative substitutions
include the substitution of one polar (hydrophilic) residue for another such
as between arginine and lysine,
between glutamine and asparagine, and between glycine and serine.
Additionally, the substitution of a basic
residue such as lysine, arginine or histidine for another, or the substitution
of one acidic residue such as aspartic
acid or glutamic acid for another acidic residue are additional examples of
conservative substitutions. Examples
of non-conservative substitutions include the substitution of a non-polar
(hydrophobic) amino acid residue such
as isoleucine, valine, leucine, alanine, methionine for a polar (hydrophilic)
residue such as cysteine, glutamine,
glutamic acid or lysine and/or a polar residue for a non-polar residue.
"Insertional variants" when referring to proteins are those with one or more
amino acids inserted
immediately adjacent to an amino acid at a particular position in a native or
starting sequence. "Immediately
adjacent" to an amino acid means connected to either the alpha-carboxy or
alpha-amino functional group of the
amino acid.
"Deletional variants," when referring to proteins, are those with one or more
amino acids in the native
or starting amino acid sequence removed. Ordinarily, deletional variants will
have one or more amino acids
deleted in a particular region of the molecule.
"Covalent derivatives," when referring to proteins, include modifications of a
native or starting protein
with an organic proteinaceous or non-proteinaceous derivatizing agent, and
post-translational modifications.
Covalent modifications are traditionally introduced by reacting targeted amino
acid residues of the protein with
an organic derivatizing agent that is capable of reacting with selected side-
chains or terminal residues, or by
harnessing mechanisms of post-translational modifications that function in
selected recombinant host cells. The
resultant covalent derivatives are useful in programs directed at identifying
residues important for biological
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activity, for immunoassays, or for the preparation of anti-protein antibodies
for immunoaffmity purification of
the recombinant glycoprotein. Such modifications are within the ordinary skill
in the art and are performed
without undue experimentation.
Certain post-translational modifications are the result of the action of
recombinant host cells on the
expressed polypeptide. Glutaminyl and asparaginyl residues are frequently post-
translationally deamidated to
the corresponding glutamyl and aspartyl residues. Alternatively, these
residues are deamidated under mildly
acidic conditions. Either form of these residues may be present in the
proteins used in accordance with the
present invention.
Other post-translational modifications include hydroxylation of proline and
lysine, phosphorylation of
hydroxyl groups of seryl or threonyl residues, methylation of the alpha-amino
groups of lysine, arginine, and
histidine side chains (T. E. Creighton, Proteins: Structure and Molecular
Properties, W.H. Freeman & Co., San
Francisco, pp. 79-86 (1983)).
Covalent derivatives specifically include fusion molecules in which proteins
of the invention are
covalently bonded to a non-proteinaceous polymer. The non-proteinaceous
polymer ordinarily is a hydrophilic
synthetic polymer, i.e. a polymer not otherwise found in nature. However,
polymers which exist in nature and
are produced by recombinant or in vitro methods are useful, as are polymers
which are isolated from nature.
Hydrophilic polyvinyl polymers fall within the scope of this invention, e.g.
polyvinylalcohol and
polyvinylpyrrolidone. Particularly useful are polyvinylalkylene ethers such a
polyethylene glycol,
polypropylene glycol. The proteins may be linked to various non-proteinaceous
polymers, such as polyethylene
glycol, polypropylene glycol or polyoxyalkylenes, in the manner set forth in
U.S. Pat. No. 4,640,835; 4,496,689;
4,301,144; 4,670,417; 4,791,192 or 4,179,337.
"Features" when referring to proteins are defined as distinct amino acid
sequence-based components of
a molecule. Features of the proteins of the present invention include surface
manifestations, local
conformational shape, folds, loops, half-loops, domains, half-domains, sites,
termini or any combination thereof
As used herein when referring to proteins the term "surface manifestation"
refers to a polypeptide
based component of a protein appearing on an outermost surface.
As used herein when referring to proteins the term "local conformational
shape" means a polypeptide
based structural manifestation of a protein which is located within a
definable space of the protein.
As used herein when referring to proteins the term "fold" means the resultant
conformation of an amino
acid sequence upon energy minimization. A fold may occur at the secondary or
tertiary level of the folding
process. Examples of secondary level folds include beta sheets and alpha
helices. Examples of tertiary folds
include domains and regions formed due to aggregation or separation of
energetic forces. Regions formed in this
way include hydrophobic and hydrophilic pockets, and the like.
As used herein the term "turn" as it relates to protein conformation means a
bend which alters the
direction of the backbone of a peptide or polypeptide and may involve one,
two, three or more amino acid
residues.
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As used herein when referring to proteins the term "loop" refers to a
structural feature of a peptide or
polypeptide which reverses the direction of the backbone of a peptide or
polypeptide and comprises four or
more amino acid residues. Oliva et al. have identified at least 5 classes of
protein loops (J. Mol Biol 266 (4):
814-830; 1997).
As used herein when referring to proteins the term "half-loop" refers to a
portion of an identified loop
having at least half the number of amino acid resides as the loop from which
it is derived. It is understood that
loops may not always contain an even number of amino acid residues. Therefore,
in those cases where a loop
contains or is identified to comprise an odd number of amino acids, a half-
loop of the odd-numbered loop will
comprise the whole number portion or next whole number portion of the loop
(number of amino acids of the
loop/2+/-0.5 amino acids). For example, a loop identified as a 7 amino acid
loop could produce half-loops of 3
amino acids or 4 amino acids (7/2=3.5+1-0.5 being 3 or 4).
As used herein when referring to proteins the term "domain" refers to a motif
of a polypeptide having
one or more identifiable structural or functional characteristics or
properties (e.g., binding capacity, serving as a
site for protein-protein interactions).
As used herein when referring to proteins the term "half-domain" means portion
of an identified
domain having at least half the number of amino acid resides as the domain
from which it is derived. It is
understood that domains may not always contain an even number of amino acid
residues. Therefore, in those
cases where a domain contains or is identified to comprise an odd number of
amino acids, a half-domain of the
odd-numbered domain will comprise the whole number portion or next whole
number portion of the domain
(number of amino acids of the domain/2+/-0.5 amino acids). For example, a
domain identified as a 7 amino acid
domain could produce half-domains of 3 amino acids or 4 amino acids (7/2=3.5+1-
0.5 being 3 or 4). It is also
understood that sub-domains may be identified within domains or half-domains,
these subdomains possessing
less than all of the structural or functional properties identified in the
domains or half domains from which they
were derived. It is also understood that the amino acids that comprise any of
the domain types herein need not
be contiguous along the backbone of the polypeptide (i.e., nonadjacent amino
acids may fold structurally to
produce a domain, half-domain or subdomain).
As used herein when referring to proteins the terms "site" as it pertains to
amino acid based
embodiments is used synonymous with "amino acid residue" and "amino acid side
chain". A site represents a
position within a peptide or polypeptide that may be modified, manipulated,
altered, derivatized or varied within
the polypeptide based molecules of the present invention.
As used herein the terms "termini or terminus" when referring to proteins
refers to an extremity of a
peptide or polypeptide. Such extremity is not limited only to the fllst or
final site of the peptide or polypeptide
but may include additional amino acids in the terminal regions. The
polypeptide based molecules of the present
invention may be characterized as having both an N-terminus (terminated by an
amino acid with a free amino
group (NH2)) and a C-terminus (terminated by an amino acid with a free
carboxyl group (COOH)). Proteins of
the invention are in some cases made up of multiple polypeptide chains brought
together by disulfide bonds or
by non-covalent forces (multimers, oligomers). These sorts of proteins will
have multiple N- and C-termini.
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Alternatively, the termini of the polypeptides may be modified such that they
begin or end, as the case may be,
with a non-polypeptide based moiety such as an organic conjugate.
Once any of the features have been identified or defined as a component of a
molecule of the invention,
any of several manipulations and/or modifications of these features may be
performed by moving, swapping,
inverting, deleting, randomizing or duplicating. Furthermore, it is understood
that manipulation of features may
result in the same outcome as a modification to the molecules of the
invention. For example, a manipulation
which involved deleting a domain would result in the alteration of the length
of a molecule just as modification
of a nucleic acid to encode less than a full length molecule would.
Modifications and manipulations can be accomplished by methods known in the
art such as site
directed mutagenesis. The resulting modified molecules may then be tested for
activity using in vitro or in vivo
assays such as those described herein or any other suitable screening assay
known in the art.
A "protein" means a polymer of amino acid residues linked together by peptide
bonds. The term, as
used herein, refers to proteins, polypeptides, and peptides of any size,
structure, or function. Typically, however,
a protein will be at least 50 amino acids long. In some instances the protein
encoded is smaller than about 50
amino acids. In this case, the polypeptide is termed a peptide. If the protein
is a short peptide, it will be at least
about 10 amino acid residues long. A protein may be naturally occurring,
recombinant, or synthetic, or any
combination of these. A protein may also comprise a fragment of a naturally
occurring protein or peptide. A
protein may be a single molecule or may be a multi-molecular complex. The term
protein may also apply to
amino acid polymers in which one or more amino acid residues is an artificial
chemical analogue of a
corresponding naturally occurring amino acid.
The term "protein expression" refers to the process by which a nucleic acid
sequence undergoes
translation such that detectable levels of the amino acid sequence or protein
are expressed.
The phrase "single-protein marker" or "single protein marker" refers to a
single protein (including all
variants of the protein) expressed by a particular cell or tissue type wherein
presence of the protein or
translational products of the gene encoding said protein, taken individually
the differential expression of such, is
indicative/predictive of a certain condition.
A "fragment of a protein," as used herein, refers to a protein that is a
portion of another protein. For
example, fragments of proteins may comprise polypeptides obtained by digesting
full-length protein isolated
from cultured cells. In one embodiment, a protein fragment comprises at least
about six amino acids. In another
embodiment, the fragment comprises at least about ten amino acids. In yet
another embodiment, the protein
fragment comprises at least about sixteen amino acids.
The terms "array" and "microarray" refer to any type of regular arrangement of
objects usually in rows
and columns. As it relates to the study of gene and/or protein expression,
arrays refer to an arrangement of
probes (often oligonucleotide or protein based) or capture agents anchored to
a surface which are used to capture
or bind to a target of interest. Targets of interest may be genes, products of
gene expression, and the like. The
type of probe (nucleic acid or protein) represented on the array is dependent
on the intended purpose of the array
(e.g., to monitor expression of human genes or proteins). The oligonucleotide-
or protein-capture agents on a
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given array may all belong to the same type, category, or group of genes or
proteins. Genes or proteins may be
considered to be of the same type if they share some common characteristics
such as species of origin (e.g.,
human, mouse, rat); disease state (e.g., cancer); structure or functions
(e.g., protein kinases, tumor suppressors);
or same biological process (e.g., apoptosis, signal transduction, cell cycle
regulation, proliferation,
differentiation). For example, one array type may be a "cancer array" in which
each of the array oligonucleotide-
or protein-capture agents correspond to a gene or protein associated with a
cancer. An "epithelial array" may be
an array of oligonucleotide- or protein-capture agents corresponding to unique
epithelial genes or proteins.
Similarly, a "cell cycle array" may be an array type in which the
oligonucleotide- or protein-capture agents
correspond to unique genes or proteins associated with the cell cycle.
The terms "immunohistochemical" or as abbreviated "IHC" as used herein refer
to the process of
detecting antigens (e.g., proteins) in a biologic sample by exploiting the
binding properties of antibodies to
antigens in said biologic sample.
The term "immunoassay" refers to a test that uses the binding of antibodies to
antigens to identify and
measure certain substances. Immunoassays often are used to diagnose disease,
and test results can provide
information about a disease that may help in planning treatment. An
immunoassay takes advantage of the
specific binding of an antibody to its antigen. Monoclonal antibodies are
often used as they usually bind only to
one site of a particular molecule, and therefore provide a more specific and
accurate test, which is less easily
confused by the presence of other molecules. The antibodies used must have a
high affinity for the antigen of
interest, because a very high proportion of the antigen must bind to the
antibody in order to ensure that the assay
has adequate sensitivity.
The term "PCR" or "RT-PCR", abbreviations for polymerase chain reaction
technologies, as used here
refer to techniques for the detection or determination of nucleic acid levels,
whether synthetic or expressed.
The term "cell type" refers to a cell from a given source (e.g., a tissue,
organ) or a cell in a given state of
differentiation, or a cell associated with a given pathology or genetic
makeup.
The term "activation" as used herein refers to any alteration of a signaling
pathway or biological
response including, for example, increases above basal levels, restoration to
basal levels from an inhibited state,
and stimulation of the pathway above basal levels.
The term "differential expression" refers to both quantitative as well as
qualitative differences in the
temporal and tissue expression patterns of a gene or a protein in diseased
tissues or cells versus normal adjacent
tissue. For example, a differentially expressed gene may have its expression
activated or completely inactivated
in normal versus disease conditions, or may be up-regulated (over-expressed)
or down-regulated (under-
expressed) in a disease condition versus a normal condition. Such a
qualitatively regulated gene may exhibit an
expression pattern within a given tissue or cell type that is detectable in
either control or disease conditions, but
is not detectable in both. Stated another way, a gene or protein is
differentially expressed when expression of the
gene or protein occurs at a higher or lower level in the diseased tissues or
cells of a patient relative to the level of
its expression in the normal (disease-free) tissues or cells of the patient
and/or control tissues or cells.
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The term "detectable" refers to an RNA expression pattern which is detectable
via the standard
techniques of polymerase chain reaction (PCR), reverse transcriptase-(RT) PCR,
differential display, and
Northern analyses, or any method which is well known to those of skill in the
art. Similarly, protein expression
patterns may be "detected" via standard techniques such as Western blots.
The term "complementary" as it relates to arrays refers to the topological
compatibility or matching
together of the interacting surfaces of a probe molecule and its target. The
target and its probe can be described
as complementary, and furthermore, the contact surface characteristics are
complementary to each other.
The term "antibody" means an immunoglobulin, whether natural or partially or
wholly synthetically
produced. All derivatives thereof that maintain specific binding ability are
also included in the term. The term
also covers any protein having a binding domain that is homologous or largely
homologous to an
immunoglobulin binding domain. An antibody may be monoclonal or polyclonal.
The antibody may be a
member of any immunoglobulin class, including any of the human classes: IgG,
IgM, IgA, IgD, and IgE, etc.
The term "antibody fragment" refers to any derivative or portion of an
antibody that is less than full-
length. In one aspect, the antibody fragment retains at least a significant
portion of the full-length antibody's
specific binding ability, specifically, as a binding partner. Examples of
antibody fragments include, but are not
limited to, Fab, Fab', F(ab')2, scFv, Fv, dsFAT diabody, and Fd fragments. The
antibody fragment may be
produced by any means. For example, the antibody fragment may be enzymatically
or chemically produced by
fragmentation of an intact antibody or it may be recombinantly produced from a
gene encoding the partial
antibody sequence. Alternatively, the antibody fragment may be wholly or
partially synthetically produced. The
antibody fragment may comprise a single chain antibody fragment. In another
embodiment, the fragment may
comprise multiple chains that are linked together, for example, by disulfide
linkages. The fragment may also
comprise a multimolecular complex. A functional antibody fragment may
typically comprise at least about 50
amino acids and more typically will comprise at least about 200 amino acids.
The term "monoclonal antibody" as used herein refers to an antibody obtained
from a population of
substantially homogeneous antibodies, i.e., the individual antibodies
comprising the population are identical
and/or bind the same epitope, except for possible variants that may arise
during production of the monoclonal
antibody, such variants generally being present in minor amounts. In contrast
to polyclonal antibody
preparations that typically include different antibodies directed against
different determinants (epitopes), each
monoclonal antibody is directed against a single determinant on the antigen.
This type of antibodies is produced
by the daughter cells of a single antibody-producing hybridoma. A monoclonal
antibody typically displays a
single binding affinity for any epitope with which it immunoreacts.
The modifier "monoclonal" indicates the character of the antibody as being
obtained from a
substantially homogeneous population of antibodies, and is not to be construed
as requiring production of the
antibody by any particular method. Monoclonal antibodies recognize only one
type of antigen The monoclonal
antibodies herein include "chimeric" antibodies (immunoglobulins) in which a
portion of the heavy and/or light
chain is identical with or homologous to corresponding sequences in antibodies
derived from a particular
species or belonging to a particular antibody class or subclass, while the
remainder of the chain(s) is identical
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with or homologous to corresponding sequences in antibodies derived from
another species or belonging to
another antibody class or subclass, as well as fragments of such antibodies.
The preparation of antibodies,
whether monoclonal or polyclonal, is know in the art. Techniques for the
production of antibodies are well
known in the art and described, e.g. in Harlow and Lane "Antibodies, A
Laboratory Manual", Cold Spring
Harbor Laboratory Press, 1988 and Harlow and Lane "Using Antibodies: A
Laboratory Manual" Cold Spring
Harbor Laboratory Press, 1999.
A monoclonal antibody may contain an antibody molecule having a plurality of
antibody combining
sites, each immunospecific for a different epitope, e.g., a bispecific
monoclonal antibody. Monoclonal
antibodies may be obtained by methods known to those skilled in the art.
Kohler and Milstein (1975), Nature,
256:495-497; U.S. Pat. No. 4,376,110; Ausubel et al. (1987, 1992), eds.,
Current Protocols in Molecular
Biology, Greene Publishing Assoc. and Wiley Inteiscience, N.Y.; Harlow and
Lane (1988), Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory; Colligan et al. (1992,
1993), eds., Current Protocols in
Immunology, Greene Publishing Assoc. and Wiley Interscience, N.Y.; Iyer et
al., Ind. .I. Med. Res., (2000),
123 :561-564.
An "antibody preparation" is meant to embrace any composition in which an
antibody may be present,
e.g., a serum (antiserum).
Antibodies may be labeled with detectable labels by one of skill in the art.
The label can be a
radioisotope, fluorescent compound, chemiluminescent compound, quantum dot,
enzyme, or enzyme co-factor,
or any other labels known in the art. In some aspects, the antibody that binds
to an entity one wishes to measure
(the primary antibody) is not labeled, but is instead detected by binding of a
labeled secondary antibody that
specifically binds to the primary antibody.
Antibodies of the invention include, but are not limited to, polyclonal,
monoclonal, multispecific,
human, humanized or chimeric antibodies, single chain antibodies, Fab
fragments, F(ab') fragments, fragments
produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies
(including, e.g., anti-Id antibodies to
antibodies of the invention), intracellularly made antibodies (i.e.,
intrabodies), and epitope-binding fragments of
any of the above. The antibodies of the invention can be from any animal
origin including birds and mammals.
Preferably, the antibodies are of human, murine (e.g., mouse and rat), donkey,
sheep, rabbit, goat, guinea pig,
camel, horse, or chicken origin.
Multispecific antibodies can be specific for different epitopes of a peptide
of the present invention, or
can be specific for both a peptide of the present invention, and a
heterologous epitope, such as a heterologous
peptide or solid support material. See, e.g., WO 93/17715; WO 92/08802; WO
91/00360; WO 92/05793; Tuff
et al., 1991,J Immunol., 147:60-69; U.S. Pat. Nos. 4,474,893; 4,714,681;
4,925,648; 5,573,920; 5,601,819; and
Kostelny et al., 1992, J. Immunol., 148:1547-1553. For example, the antibodies
may be produced against a
peptide containing repeated units of a FAS peptide sequence of the invention,
or they may be produced against a
peptide containing two or more FAS peptide sequences of the invention, or the
combination thereof
Moreover, antibodies can also be prepared from any region of the FAS peptides
of the invention. In
addition, if a polypeptide is a receptor protein, antibodies can be developed
against an entire receptor or portions
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of the receptor, for example, an intracellular domain, an extracellular
domain, the entire transmembrane domain,
specific transmembrane segments, any of the intracellular or extracellular
loops, or any portions of these regions.
Antibodies can also be developed against specific functional sites, such as
the site of ligand binding, or sites that
are glycosylated, phosphorylated, mpistylated, or amidated, for example.
By "amplification" is meant production of multiple copies of a target nucleic
acid that contains at least
a portion of an intended specific target nucleic acid sequence. The multiple
copies may be referred to as
amplicons or amplification products. Preferably, the amplified target contains
less than the complete target gene
sequence (introns and exons) or an expressed target gene sequence (spliced
transcript of exons and flanking
untranslated sequences). For example, FAS-specific amplicons may be produced
by amplifying a portion of the
FAS target polynucleotide by using amplification primers which hybridize to,
and initiate polymerization from,
internal positions of the FAS target polynucleotide. Preferably, the amplified
portion contains a detectable target
sequence which may be detected using any of a variety of well known methods.
By "primer" or "amplification primer" is meant an oligonucleotide capable of
binding to a region of a
target nucleic acid or its complement and promoting nucleic acid amplification
of the target nucleic acid. In
most cases a primer will have a free 3' end which can be extended by a nucleic
acid polymerase. All
amplification primers include a base sequence capable of hybridizing via
complementary base interactions
either directly with at least one strand of the target nucleic acid or with a
strand that is complementary to the
target sequence. Amplification primers serve as substrates for enzymatic
activity that produces a longer nucleic
acid product.
A "target-binding sequence" of an amplification primer is the portion that
determines target specificity
because that portion is capable of annealing to a target nucleic acid strand
or its complementary strand. The
complementary target sequence to which the target-binding sequence hybridizes
is referred to as a primer-
binding sequence.
By "detecting" an amplification product is meant any of a variety of methods
for determining the
presence of an amplified nucleic acid, such as, for example, hybridizing a
labeled probe to a portion of the
amplified product. A labeled probe is an oligonucleotide that specifically
binds to another sequence and contains
a detectable group which may be, for example, a fluorescent moiety, a
chemiluminescent moiety, a radioisotope,
biotin, avidin, enzyme, enzyme substrate, or other reactive group.
By "nucleic acid amplification conditions" is meant environmental conditions
including salt
concentration, temperature, the presence or absence of temperature cycling,
the presence of a nucleic acid
polymerase, nucleoside triphosphates, and cofactors which are sufficient to
permit the production of multiple
copies of a target nucleic acid or its complementary strand using a nucleic
acid amplification method. Many
well-known methods of nucleic acid amplification require thermocycling to
alternately denature double-
stranded nucleic acids and hybridize primers.
The term "biomarkef ' as used herein refers to a characteristic that is
objectively measured and
evaluated as an indicator of normal biological processes, pathogenic processes
or biological responses to a
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therapeutic intervention. They can suggest etiology of, susceptibility to,
activity of or progress of a disease
substance indicative of a biological state.
The term "biological sample" or "biologic sample" refers to a sample obtained
from an organism (e.g.,
a human patient) or from components (e.g., cells) or from body fluids (e.g.,
blood, serum, sputum, urine, etc) of
an organism. The sample may be of any biological tissue, organ, organ system
or fluid. The sample may be a
"clinical sample" which is a sample derived from a patient. Such samples
include, but are not limited to, sputum,
blood, blood cells (e.g., white cells), amniotic fluid, plasma, semen, bone
marrow, and tissue or core, fine or
punch needle biopsy samples, aspirations, urine, peritoneal fluid, and pleural
fluid, or cells therefrom. Biological
samples may also include sections of tissues such as frozen sections taken for
histological purposes. A biological
sample may also be referred to as a "patient sample."
The term "condition" refers to the status of any cell, organ, organ system or
organism. Conditions may
reflect a disease state or simply the physiologic presentation or situation of
an entity. Conditions may be
characterized as phenotypic conditions such as the macroscopic presentation of
a disease or genotypic
conditions such as the underlying gene or protein expression profiles
associated with the condition. Conditions
may be benign or malignant.
The term "cancer" in an individual refers to the presence of cells possessing
characteristics typical of
cancer-causing cells, such as uncontrolled proliferation, immortality,
metastatic potential, rapid growth and
proliferation rate, and certain characteristic morphological features. Often,
cancer cells will be in the form of a
tumor, but such cells may exist alone within an individual, or may circulate
in the blood stream as independent
cells, such as leukemic cells.
The term "breast cancer" means a cancer of the breast tissue or associated
lymph nodes.
The term "cell growth" is principally associated with growth in cell numbers,
which occurs by means
of cell reproduction (i.e. proliferation) when the rate of the latter is
greater than the rate of cell death (e.g. by
apoptosis or necrosis), to produce an increase in the size of a population of
cells, although a small component of
that growth may in certain circumstances be due also to an increase in cell
size or cytoplasmic volume of
individual cells. An agent that inhibits cell growth can thus do so by either
inhibiting proliferation or stimulating
cell death, or both, such that the equilibrium between these two opposing
processes is altered.
The term "tumor growth" or "tumor metastases growth", as used herein, unless
otherwise indicated, is
used as commonly used in oncology, where the term is principally associated
with an increased mass or volume
of the tumor or tumor metastases, primarily as a result of tumor cell growth.
The term "metastasis" means the process by which cancer spreads from the place
at which it first arose
as a primary tumor to distant locations in the body. Metastasis also refers to
cancers resulting from the spread of
the primary tumor. For example, someone with breast cancer may show metastases
in their lymph system,
liver, bones or lungs.
The term "lesion" or "lesion site" as used herein refers to any abnormal,
generally localized, structural
change in a bodily part or tissue. Calcifications or fibrocystic features are
examples of lesions of the present
invention.
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The term "clinical management parameter" refers to a metric or variable
considered important in the
detecting, screening, diagnosing, staging or stratifying patients, or
determining the progression of,
regression of and/or survival from a disease or condition. Examples of such
clinical management
parameters include, but are not limited to survival in years, disease related
death, early or late recurrence,
degree of regression, metastasis, responsiveness to treatment, effectiveness
of treatment or the likelihood of
progression to breast cancer.
The term "endpoint" means a final stage or occurrence along a path or
progression.
The term "tumor assessment endpoint" means an endpoint observation or
calculation based on the
stage, status or occurrence of a tumor. Examples of endpoints based on tumor
assessments include, but are not
limited to, survival, disease free survival (DFS), objective response rate
(ORR), time to progression (TTP),
progression free survival (PFS), and time to treatment failure (1'1F).
The phrase "morphologic prognosis parameter or feature" means a feature of the
cancerous phenotype
used to predict an outcome. Morphologic prognosis parameters or features
include axillary lymph node
metastasis (which is the most significant), tumor type, tumor grade, and tumor
size. Secondary but important
morphologic parameters also considered predictive include the extent of an
intraductal component in patients
with mixed intraductal and infiltrating ductal carcinoma, proven
intralymphatic and intravascular invasion, and
high mitotic index.
The phrase "lymph node negative" as used herein refers to the status of a
patient where at least one or
more removed or biopsied lymph nodes showed no evidence of metastatic
carcinoma. In one
embodiment, a lymph node negative status is defined as the situation where
more than 4, more than 5
or more than 6 removed or biopsied lymph nodes showed no evidence of
metastatic carcinoma.
The term "treating" as used herein, unless otherwise indicated, means
reversing, alleviating, inhibiting
the progress of, or preventing, either partially or completely, the growth of
tumors, tumor metastases, or other
cancer-causing or neoplastic cells in a patient with cancer. The term
"treatment" as used herein, unless otherwise
indicated, refers to the act of treating.
The phrase "a method of treating" or its equivalent, when applied to, for
example, cancer refers to a
procedure or course of action that is designed to reduce, eliminate or prevent
the number of cancer cells in an
individual, or to alleviate the symptoms of a cancer. "A method of treating"
cancer or another proliferative
disorder does not necessarily mean that the cancer cells or other disorder
will, in fact, be completely eliminated,
that the number of cells or disorder will, in fact, be reduced, or that the
symptoms of a cancer or other disorder
will, in fact, be alleviated. Often, a method of treating cancer will be
performed even with a low likelihood of
success, but which, given the medical history and estimated survival
expectancy of an individual, is nevertheless
deemed an overall beneficial course of action.
The term "predicting" means a statement or claim that a particular event will,
or is very likely to, occur
in the future.
The term "prognosing" means a statement or claim that a particular biologic
event will, or is very likely
to, occur in the future.
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The term "progression" or "cancer progression" means the advancement or
worsening of or toward a
disease or condition.
The term "regression" or "degree of regression" refers to the reversal, either
phenotypically or
genotypically, of a cancer progression. Slowing or stopping cancer progression
may be considered regression.
The term "stratifying" as it relates to patients means the parsing of patients
into groups of predicted
outcomes along a continuum of from a positive outcome (such as disease free)
to moderate or good outcomes
(such as improved quality of life or increased survival) to poor outcomes
(such as terminal prognosis or death).
The term "therapeutically effective agent" means a composition that will
elicit the biological or
medical response of a tissue, organ, system, organism, animal or human that is
being sought by the researcher,
veterinarian, medical doctor or other clinician.
The term "therapeutically effective amount" or "effective amount" means the
amount of the subject
compound or combination that will elicit the biological or medical response of
a tissue, organ, system,
organism, animal or human that is being sought by the researcher,
veterinarian, medical doctor or other
clinician.
The term "correlate" or "correlation" as used herein refers to a relationship
between two or more
random variables or observed data values. A correlation may be statistical if,
upon analysis by statistical means
or tests, the relationship is found to satisfy the threshold of significance
of the statistical test used.
Clinical Management Parameters
The invention relates to compositions, methods and assays for detecting,
screening for, or
diagnosing breast cancer; staging or stratifying breast cancer patients; and
determining the
progression of, regression of and/or survival from breast cancer.
In doing so, the present invention provides methods, algorithms and other
clinical tools to
augment traditional diagnostic, prognostic and/or therapeutic paradigms.
Combination approaches
using one or more biomarkers in the determination of the value of one or more
clinical management
parameters also are envisioned. For example, methods of this invention that
measure both FAS and any
one or more biomarkers can provide potentially superior results to diagnostic
assays measuring just one of these
biomarkers absent the measurement of FAS. FAS may even be measured as the sole
marker. This dual, or
multi-biomarker approach, in combination with imaging techniques provides even
further superiority. Any dual,
or multiple, biomarker approach (with or without companion imaging) thus
reduces the number of patients that
are predicted not to benefit from treatment, and thus potentially reduces the
number of patients that fail to
receive treatment that may extend their lives significantly.
Clinical management parameters addressed by the present invention include
survival in years, disease
related death, early or late recurrence, degree of regression, metastasis,
responsiveness to treatment,
effectiveness of treatment.
Believing that FAS expression is a superior predictor of many of the clinical
management
parameters important to clinicians treating patients having or suspected of
having breast cancer, the
present invention involves the rapid identification of FAS expression in
tissue, cells and/or serum.
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The method generally comprises the following steps: (a) obtaining a biological
sample from a cancer
patient; (b) contacting the sample with a detection agent specific for FAS;
(c) detecting the presence, amount or
levels of FAS in (b); and (d) correlating the presence, amount or levels of
FAS (alone or in combination) with
the one or more clinical management parameters in order to aid in the
prevention, diagnosis or treatment of
breast cancer.
The biological sample may be cells or tissue, and preferably is serum or
plasma containing cells.
However, the cells also may be obtained from tissue samples or cell cultures
such as in ex vivo or in situ
methods.
The detection agent may a nucleic acid probe specific for FAS, or an anti-FAS
antibody.
FAS Probes
The present invention provides an assay method comprising novel nucleic acid
based probes useful in
the detection of the FAS gene or protein in a biological sample. The sample
may be breast tissue or non-breast
tissue. The non-breast tissue can include, for example, blood, lymph node,
breast or breast cyst, nipple
aspirations, kidney, liver, lung, muscle, stomach or intestinal tissue.
The present invention also includes a method for detecting and quantifying the
FAS-specific RNA
species. Other embodiments of the invention include methods for detecting
other biomarker species,
individually or in combination with each other or FAS sequences. Moreover,
detection of these markers
individually and in combination, are clinically important because cancers from
individual patients may express
one or more of the markers, such that detecting one or more of the markers
decreases the potential of false
negatives during diagnosis that might otherwise result if the presence of only
one marker was tested.
In situ hybridization (ISH) and fluorescence in situ hybridi7ation (FISH)
The present invention provides methods of detecting target nucleic acids via
in situ
hybridization and fluorescent in situ hybridization using novel probes. The
methods of in situ
hybridization were first developed in 1969 and many improvements have been
made since. The basic
technique utilizes hybridization kinetics for RNA and/or DNA via hydrogen
bonding. By labeling
sequences of DNA or RNA of sufficient length (approximately 50-300 base
pairs), selective probes can
be made to detect particular sequences of DNA or RNA. The application of these
probes to tissue
sections allows DNA or RNA to be localized within tissue regions and cell
types. Methods of probe
design are known to those of skill in the art. Detection of hybridized probe
and target may be performed
in several ways known in the art. Most prominently is through the use of
detection labels attached to the
probes. Probes of the present invention may be single or double stranded and
may be DNA, RNA, or
mixtures of DNA and RNA. They may also constitute any nucleic acid based
construct. Labels for the
probes of the present invention may be radioactive or non-radioactive and the
design and use of such
labels is well known in the art.
FAS Antibodies
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In one embodiment, the present invention utilizes anti-FAS antibodies in an
ELISA assay.
The anti-FAS antibodies preferably are those disclosed in PCT Publication
PCT/US2010/030545
published October 14, 2010, and PCT/US2010/046773 published March 17, 2011,
respectively.
The antibodies used in the present invention for detection or capture of FAS
are novel anti-FAS
antibodies that are highly specific for human FAS.
In one embodiment, commercial antibodies for the detection of FAS are used.
For IHC the antibodies
which may be used are the human anti-FASN Antibody, Affinity Purified (Catalog
No. A301-324A)
from Bethyl Laboratories (Montgomery, TX) and for ELISA studies, antibodies
which may be used
include the Fatty Acid Synthase Antibody Pair (Catalog No. H00002194-AP11)
from Novus
Biologicals (Littleton, CO). The pair contains a Capture antibody which is
rabbit affinity purified
polyclonal anti-FASN (100 ug) and a Detection antibody which is mouse
monoclonal anti-FASN,
IgG1 Kappa (20 ug).
In one embodiment, the present antibodies are monoclonal antibodies specific
for a human FAS
sequence selected from SEQ ID NOs. 1-5 (Table 1). FAS peptides are derived
from the protein encoded by the
FAS (Fatty Acid Synthase) gene; GenBank NM_004104; SEQ ID NO: 6.
In another embodiment, the present antibodies are used as capture antibodies
in a sandwich ELISA
assay.
Table 1: FAS Peptides
Hybridoma FAS Peptide SEQ ID
A VAQGQWEPSGXAP 1
B PSGPAPTNXGALE 2
C TLEQQHXVAQGQW 3
D EVDPGSAELQKVLQGD 4
E ELSSKADEASELAC 5
FAS Antibodies and Detection Rate
In one embodiment, the FAS antibodies disclosed herein may be used in the
detection of breast cancer,
either alone or in combination with measurements of other biomarkers.
Measurements may be made for
example, in tissue, cells, serum or plasma of patients.
Gene Expression and Localization of Expression
In one embodiment of the invention, FAS expression is measured relative to the
expression of
one or more additional genes and/or at one or more different biopsy sites.
Comparisons of gene
expression within the cancer site as compared to expression at the margin of
the cancer and at sites
distal from the cancer allow conclusions to be drawn about the status of a
sample and whether it will
become cancerous. These conclusions then allow for improved predictions about
metastasis and
consequently survival. Additional patient parameters also may be combined with
the gene expression
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data to improve the predictive power of the method.
FAS and Degree of Regression
In one embodiment, FAS expression levels are used as a predictor of the
probability of cancer
regression which allows stratification between POOR and GOOD outcomes for
individual patients. In
this method, FAS expression is correlated with degree of regression where
higher FAS expression
levels are predictive of clinical outcomes. It has been determined that FAS
expression level is an
excellent predictor of both GOOD and POOR outcomes.
Assays and Kits
Any of the compositions described herein may be comprised in a kit. In one
embodiment, antibodies to
one or more of the expression products of the FAS genes disclosed herein are
included. Antibodies may be
included to provide concentrations of from about 0.1 iLig/mL to about 500
lag/mL, from about 0.1 iLig/mL to
about 50 iLig/mL or from about 1 iLig/mL to about 5 iLig/mL or any value
within the stated ranges. The kit may
further include reagents or instructions for creating or synthesizing further
probes, labels or capture agents. It
may also include one or more buffers, such as a nuclease buffer, transcription
buffer, or a hybridization buffer,
compounds for preparing a DNA template, cDNA, primers, probes or label, and
components for isolating any
of the foregoing. Other kits of the invention may include components for
making a nucleic acid or peptide array
including all reagents, buffers and the like and thus, may include, for
example, a solid support.
The components of the kits may be packaged either in aqueous media or in
lyophilized form. The
container means of the kits will generally include at least one vial, test
tube, flask, bottle, syringe or other
container means, into which a component may be placed, and preferably,
suitably aliquoted. Where there are
more than one component in the kit (labeling reagent and label may be packaged
together), the kit also will
generally contain a second, third or other additional container into which the
additional components may be
separately placed. However, various combinations of components may be
comprised in a vial or similar
container. The kits of the present invention also will typically include a
means for containing the detection
reagents, e.g., nucleic acids or proteins or antibodies, and any other reagent
containers in close confinement for
commercial sale. Such containers may include injection or blow-molded plastic
containers into which the
desired vials are retained.
When the components of the kit are provided in one and/or more liquid
solutions, the liquid solution is
an aqueous solution, with a sterile aqueous solution being particularly
preferred. However, the components of
the kit may be provided as dried powder(s). When reagents and/or components
are provided as a dry powder,
the powder can be reconstituted by the addition of a suitable solvent. It is
envisioned that the solvent may also be
provided in another container means. In some embodiments, labeling dyes are
provided as a dried power. It is
contemplated that 10,20, 30,40, 50, 60, 70, 80,90, 100, 120, 120, 130, 140,
150, 160, 170, 180, 190, 200, 300,
400, 500, 600, 700, 800, 900, 1000 micrograms or at least or at most those
amounts of dried dye are provided in
kits of the invention. The dye may be re-suspended in any suitable solvent,
such as DMSO.
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Kits may also include components that preserve or maintain the compositions
that protect against their
degradation. Such kits generally will comprise, in suitable means, distinct
containers for each individual reagent
or solution.
Certain assay methods of the invention comprises contacting a tissue sample
from an individual with a
group of antibodies specific for some or all of the genes or proteins
disclosed, and determining the occurrence of
up- or down-regulation of these genes or proteins in the sample. The use of
TMAs allows numerous samples,
including control samples, to be assayed simultaneously.
The method preferably also includes detecting and/or quantitating control or
"reference proteins".
Detecting and/or quantitating the reference proteins in the samples normalizes
the results and thus provides
further assurance that the assay is working properly. In a currently preferred
embodiment, antibodies specific
for one or more of the following reference proteins are included: beta-actin
(ACTB), glyceraldehyde-3-
phosphate dehydrogenase (GAPDH), beta glucoronidase (GUSB) as positive
controls while negative controls
include large ribosomal protein (RPLPO) and/or transferrin receptor (TRFC).
Beta actin may be used as the
positive control for WIC.
The present invention further comprises a kit containing reagents for
conducting an WIC analysis of
tissue samples or cells from individuals, e.g., patients, including antibodies
specific for one or more proteins and
for any reference proteins. The antibodies are preferably tagged with means
for detecting the binding of the
antibodies to the proteins of interest, e.g., detectable labels. Preferred
detectable labels include fluorescent
compounds or quantum dots; however other types of detectable labels may be
used. Detectable labels for
antibodies are commercially available, e.g. from Ventana Medical Systems, Inc.
Immunohistochemical methods for detecting and quantitating protein expression
in tissue samples are
well known. Any method that permits the determination of expression of several
different proteins can be used.
Such methods can be efficiently carried out using automated instruments
designed for immunohistochemical
(IHC) analysis. Instruments for rapidly performing such assays are
commercially available, e.g., from Ventana
Molecular Discovery Systems or Lab Vision Corporation. Methods according to
the present invention using
such instruments are carried out according to the manufacturer's instructions.
Protein-specific antibodies for use in such methods or assays are readily
available or can be prepared
using well-established techniques. Antibodies specific for the proteins herein
can be obtained, for example,
from Cell Signaling Technology, Inc, Santa Cruz Biotechnology, Inc. or Abcam.
ImmunoAssays
The present invention provides for new assays useful in the diagnosis,
prognosis and prediction of
breast cancer and the elucidation of clinical management parameters associated
with breast cancer. The
immunoassays of the present invention utilize the anti-FAS polyclonal or
monoclonal antibodies described
herein to specifically bind to FAS in a biological sample. Any type of
immunoassay format may be used,
including, without limitation, enzyme immunoassays (EIA, ELISA),
radioimmunoassay (RIA),
fluoroimmunoassay (HA), chemiluminescent immunoassay (CLIA), counting
immunoassay (CIA),
immunohistochemistry (IHC), agglutination, nephelometry, turbidimetry or
Western Blot. These and other
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types of immunoassays are well-known and are described in the literature, for
example, in Immunochemistry,
Van Oss and Van Regenmortel (Eds), CRC Press, 1994; The Immunoassay Handbook,
D. Wild (Ed.), Elsevier
Ltd., 2005; and the references disclosed therein.
The preferred assay format for the present invention is the enzyme-linked
immunosorbent assay
(ELISA) format. ELISA is a highly sensitive technique for detecting and
measuring antigens or antibodies in a
solution in which the solution is run over a surface to which immobilized
antibodies specific to the substance
have been attached, and if the substance is present, it will bind to the
antibody layer, and its presence is verified
and visualized with an application of antibodies that have been tagged or
labeled so as to permit detection.
ELISAs combine the high specificity of antibodies with the high sensitivity of
enzyme assays by using
antibodies or antigens coupled to an easily assayed enzyme that possesses a
high turnover number such as
alkaline phosphatase (AP) or horseradish peroxidase (HRP), and are very useful
tools both for determining
antibody concentrations (antibody titer) in sera as well as for detecting the
presence of antigen.
There are many different types of ELISAs; the most common types include
"direct ELISA," "indirect
ELISA," "sandwich ELISA" and cell-based ELISA (C-ELISA). Performing an ELISA
involves at least one
antibody with specificity for a particular antigen. The sample with an unknown
amount of antigen is
immobilized on a solid support (usually a polystyrene microtiter plate) either
non-specifically (via adsorption to
the surface) or specifically (via capture by another antibody specific to the
same antigen, in a "sandwich"
ELISA). After the antigen is immobilized the detection antibody is added,
forming a complex with the antigen.
The detection antibody can be covalently linked to an enzyme, or can itself be
detected by a secondary antibody
which is linked to an enzyme through bioconjugation. Between each step the
plate typically is washed with a
mild detergent solution to remove any proteins or antibodies that are not
specifically bound. After the final wash
step the plate is developed by adding an enzymatic substrate tagged with a
detectable label to produce a visible
signal, which indicates the quantity of antigen in the sample.
In a typical microtiter plate sandwich immunoassay, an antibody ("capture
antibody") is adsorbed or
immobilized onto a substrate, such as a microtiter plate. Monoclonal
antibodies are preferred as capture
antibodies due to their greater specificity, but polyclonal antibodies also
may be used. When the test sample is
added to the plate, the antibody on the plate will bind the target antigen
from the sample, and retain it in the
plate. When a second antibody ("detection antibody") or antibody pair is added
in the next step, it also binds to
the target antigen (already bound to the monoclonal antibody on the plate),
thereby forming an antigen
'sandwich' between the two different antibodies.
This binding reaction can then be measured by radio-isotopes, as in a radio-
immunoassay format
(RIA); by enzymes, as in an enzyme immunoassay format (EIA or ELISA); or other
detectable label, attached
to the detection antibody. The label generates a color signal proportional to
the amount of target antigen present
in the original sample added to the plate. Depending on the immunoassay
format, the degree of color can be
detected and measured with the naked eye (as with a home pregnancy test), a
scintillation counter (for an RIA),
or with a spectrophotometric plate reader (for an EIA or ELISA).
The assay then is carried out according to the following general steps:
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Step 1: Capture antibodies are adsorbed onto the well of a plastic microtiter
plate (no sample added);
Step 2: A test sample (such as human serum) is added to the well of the plate,
under conditions
sufficient to permit binding of the target antigen to the capture antibody
already bound to the plate, thereby
retaining the antigen in the well;
Step 3: Binding of a detection antibody or antibody pair (with enzyme or other
detectable moiety
attached) to the target antigen (already bound to the capture antibody on the
plate), thereby forming an antigen
"sandwich" between the two different antibodies. The detectable label on the
detection antibodies will generate
a color signal proportional to the amount of target antigen present in the
original sample added to the plate.
In an alternative embodiment, sometimes referred to as an antigen-down
immunoassay, the analyte
(rather than an antibody) is coated onto a substrate, such as a microtiter
plate, and used to bind antibodies found
in a sample. When the sample is added (such as human serum), the antigen on
the plate is bound by antibodies
(IgE for example) from the sample, which are then retained in the well. A
species-specific antibody (anti-
human IgE for example) labeled with an enzyme such as horse radish peroxidase
(HRP) is added next, which,
binds to the antibody bound to the antigen on the plate. The higher the
signal, the more antibodies there are in
the sample.
In another embodiment, an immunoassay may be structured in a competitive
inhibition format.
Competitive inhibition assays are often used to measure small analytes because
competitive inhibition assays
only require the binding of one antibody rather than two as is used in
standard ELISA formats. In a sequential
competitive inhibition assay, the sample and conjugated analyte are added in
steps similar to a sandwich assay,
while in a classic competitive inhibition assay, these reagents are incubated
together at the same time.
In a typical sequential competitive inhibition assay format, a capture
antibody is coated onto a
substrate, such as a microtiter plate. When the sample is added, the capture
antibody captures free analyte out of
the sample. In the next step, a known amount of analyte labeled with a
detectable label, such as an enzyme or
enzyme substrate, added. The labeled analyte also attempts to bind to the
capture antibody adsorbed onto the
plate, however, the labeled analyte is inhibited from binding to the capture
antibody by the presence of
previously bound analyte from the sample. This means that the labeled analyte
will not be bound by the
monoclonal on the plate if the monoclonal has already bound unlabeled analyte
from the sample. The amount
of unlabeled analyte in the sample is inversely proportional to the signal
generated by the labeled analyte. The
lower the signal, the more unlabeled analyte there is in the sample. A
standard curve can be constructed using
serial dilutions of an unlabeled analyte standard. Subsequent sample values
can then be read off the standard
curve as is done in the sandwich ELISA formats. The classic competitive
inhibition assay format requires the
simultaneous addition of labeled (conjugated analyte) and unlabeled analyte
(from the sample). Both labeled
and unlabeled analyte then compete simultaneously for the binding site on the
monoclonal capture antibody on
the plate. Like the sequential competitive inhibition format, the colored
signal is inversely proportional to the
concentration of unlabeled target analyte in the sample. Detection of labeled
analyte can be read on a microtiter
plate reader.
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In addition to microtiter plates, immunoassays are also may be configured as
rapid tests, such as a
home pregnancy test. Like microtiter plate assays, rapid tests use antibodies
to react with antigens and can be
developed as sandwich formats, competitive inhibition formats, and antigen-
down formats. With a rapid test,
the antibody and antigen reagents are bound to porous membranes, which react
with positive samples while
channeling excess fluids to a non-reactive part of the membrane. Rapid
immunoassays commonly come in two
configurations: a lateral flow test where the sample is simply placed in a
well and the results are read
immediately; and a flow through system, which requires placing the sample in a
well, washing the well, and
then finally adding an analyte-detectable label conjugate and the result is
read after a few minutes. One sample
is tested per strip or cassette. Rapid tests are faster than microtiter plate
assays, require little sample processing,
are often cheaper, and generate yes/no answers without using an instrument.
However, rapid immunoassays are
not as sensitive as plate-based immunoassays, nor can they be used to
accurately quantitate an analyte.
The preferred technique for use in the present invention to detect the amount
of FAS in circulating cells
is the sandwich ELISA, in which highly specific monoclonal antibodies are used
to detect sample antigen. The
sandwich ELISA method comprises the following general steps:
1. Prepare a surface to which a known quantity of capture antibody is
bound;
2. (Optionally) block any non specific binding sites on the surface;
3. Apply the antigen-containing sample to the surface;
4. Wash the surface, so that unbound antigen is removed;
5. Apply primary (detection) antibodies that bind specifically to the bound
antigen;
6. Apply enzyme-linked secondary antibodies which are specific to the
primary antibodies;
7. Wash the plate, so that the unbound antibody-enzyme conjugates are
removed;
8. Apply a chemical which is converted by the enzyme into a detectable
(e.g., color or
fluorescent or electrochemical) signal; and
9. Measure the absorbance or fluorescence or electrochemical signal to
determine the presence
and quantity of antigen.
In an alternate embodiment, the primary antibody (step 5) is linked to an
enzyme; in this embodiment,
the use of a secondary antibody conjugated to an enzyme (step 6) is not
necessary if the primary antibody is
conjugated to an enzyme. However, use of a secondary-antibody conjugate avoids
the expensive process of
creating enzyme-linked antibodies for every antigen one might want to detect.
By using an enzyme-linked
antibody that binds the Fc region of other antibodies, this same enzyme-linked
antibody can be used in a variety
of situations. The major advantage of a sandwich ELISA is the ability to use
crude or impure samples and still
selectively bind any antigen that may be present. Without the first layer of
"capture" antibody, any proteins in
the sample (including serum proteins) may competitively adsorb to the plate
surface, lowering the quantity of
antigen immobilized.
In one embodiment of the present invention, a solid phase substrate, such as a
microtiter plate or strip,
is treated in order to fix or immobilize a capture antibody to the surface of
the substrate. The material of the
solid phase is not particularly limited as long as it is a material of a usual
solid phase used in immunoassays.
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Examples of such material include polymer materials such as latex, rubber,
polyethylene, polypropylene,
polystyrene, a styrene-butadiene copolymer, polyvinyl chloride, polyvinyl
acetate, polyacrylamide,
polymethacrylate, a styrene-methacrylate copolymer, polyglycidyl methacrylate,
an acrolein-ethyleneglycol
dimethacrylate copolymer, polyvinylidene difluoride (PVDF), and silicone;
agarose; gelatin; red blood cells;
and inorganic materials such as silica gel, glass, inert alumina, and magnetic
substances. These materials may be
used singly or in combination of two or more thereof
The form of the solid phase is not particularly limited insofar as the solid
phase is in the form of a usual
solid phase used in immunoassays, for example in the form of a microtiter
plate, a test tube, beads, particles, and
nanoparticles. The particles include magnetic particles, hydrophobic particles
such as polystyrene latex,
copolymer latex particles having hydrophilic groups such as an amino group and
a carboxyl group on the
surfaces of the particles, red blood cells and gelatin particles. The solid
phase is preferably a microtiter plate or
strip, such as those available from Cell Signalling Technology, Inc.
The capture antibody preferably is one or more monoclonal anti-FAS antibodies
described herein that
specifically bind to at least a portion of one or more of the peptide
sequences of SEQ ID NO. 1-5. Where
microtiter plates or strips are used, the capture antibody is immobilized
within the wells. Techniques for coating
and/or immobilizing proteins to solid phase substrates are known in the art,
and can be achieved, for example,
by a physical adsorption method, a covalent bonding method, an ionic bonding
method, or a combination
thereof. See, e.g., W. Luttmann et al., Immunology, Ch. 4.3.1 (pp. 92-94),
Elsevier, Inc. (2006) and the
references cited therein. For example, when the binding substance is avidin or
streptavidin, a solid phase to
which biotin was bound can be used to fix avidin or streptavidin to the solid
phase. The amounts of the capture
antibody, the detection antibody and the solid phase to be used can also be
suitably established depending on the
antigen to be measured, the antibody to be used, and the type of the solid
phase or the like. Protocols for coating
microtiter plates with capture antibodies, including tools and methods for
calculating the quantity of capture
antibody, are described for example, on the websites for Immunochemistry
Technologies, LLC (Bloomington,
MN) and Meso Scale Diagnostics, LLC (Gaithersburg, MD).
The detection antibody can be any anti-FAS antibody. Anti-FAS antibodies are
commercially
available, for example, from Cell Signaling Technologies, Inc., Santa Cruz
Biotechnology, EMD Biosciences,
and others. The detection antibody also may be an anti-FAS antibody as
disclosed herein that is specific for one
or more of SEQ ID NOs. 1-5. In one embodiment, the detection antibody may be
directly conjugated with a
detectable label, or an enzyme. If the detection antibody is not conjugated
with a detectable label or an enzyme,
then a labeled secondary antibody that specifically binds to the detection
antibody is included. Such detection
antibody "pairs" are commercially available, for example, from Cell Signaling
Technologies, Inc.
Techniques for labeling antibodies with detectable labels are well-established
in the art. As used
herein, the term "detectable label" refers to a composition detectable by
spectroscopic, photochemical,
biochemical, immunochemical, or chemical means. The detectable label can be
selected, e.g., from a group
consisting of radioisotopes, fluorescent compounds, chemiluminescent
compounds, enzymes, and enzyme co-
factors, or any other labels known in the art. See, e.g., Zola, Monoclonal
Antibodies: A Manual of Techniques,
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pp. 147-158 (CRC Press, Inc. 1987). A detectable label can be attached to the
subject antibodies and is selected
so as to meet the needs of various uses of the method which are often dictated
by the availability of assay
equipment and compatible immunoassay procedures. Appropriate labels include,
without limitation,
radionuclides, enzymes (e.g., alkaline phosphatase, horseradish peroxidase,
luciferase, or 13-glactosidase),
fluorescent moieties or proteins (e.g., fluorescein, rhodamine, phycoerythrin,
GFP, or BFP), or luminescent
moieties (e.g., EvidotO quantum dots supplied by Evident Technologies, Troy,
NY, or QdotTM nanoparticles
supplied by the Quantum Dot Corporation, Palo Alto, Calif).
Preferably, the sandwich immunoassay of the present invention comprises the
step of measuring the
labeled secondary antibody, which is bound to the detection antibody, after
formation of the capture antibody-
antigen-detection antibody complex on the solid phase. The method of measuring
the labeling substance can be
appropriately selected depending on the type of the labeling substance. For
example, when the labeling
substance is a radioisotope, a method of measuring radioactivity by using a
conventionally known apparatus
such as a scintillation counter can be used. When the labeling substance is a
fluorescent substance, a method of
measuring fluorescence by using a conventionally known apparatus such as a
luminometer can be used.
When the labeling substance is an enzyme, a method of measuring luminescence
or coloration by
reacting an enzyme substrate with the enzyme can be used. The substrate that
can be used for the enzyme
includes a conventionally known luminescent substrate, calorimetric substrate,
or the like. When an alkaline
phosphatase is used as the enzyme, its substrate includes chemilumigenic
substrates such as CDP-star0 (4-
chloro-3-(methoxyspiro (1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.1.-
sup.3.7]decane)-4-yl)disodium
phenylphosphate) and CSPDO (3-(4-methoxyspiro(1,2-dioxetane-3,2-(5'-
chloro)tricyclo[3.3.1.1<sup>3</sup>.7]-
decane)-4-yl)disodium phenylphosphate) and colorimetric substrates such as p-
nitrophenyl phosphate, 5-bromo-
4-chloro-3-indolyl-phosphoric acid (BCIP), 4-nitro blue tetrazolium chloride
(NBT), and iodonitro tetrazolium
(INT). These luminescent or calorimetric substrates can be detected by a
conventionally known
spectrophotometer, luminometer, or the like.
In one embodiment, the detectable labels comprise quantum dots (e.g., EvidotO
quantum dots supplied
by Evident Technologies, Troy, NY, or QdotTM nanoparticles supplied by the
Quantum Dot Corporation, Palo
Alto, Calif.). Techniques for labeling proteins, including antibodies, with
quantum dots are known. See, e.g.,
Goldman et al., Phys. Stat. Sol., 229(1): 407-414 (2002); Zdobnova et al., J.
Biomed. Opt., 14(2):021004 (2009);
Lao et al., JACS, 128(46):14756-14757 (2006); Mattoussi et al., JACS,
122(49):12142-12150 (2000); and
Mason et al., Methods in Molecular Biology: NanoBiotechnology Protocols,
303:35-50 (Springer Protocols,
2005). Quantum-dot antibody labeling kits are commercially available, e.g.,
from Invitrogen (Carlsbad, CA)
and Millipore (Billerica, MA).
The sandwich immunoassay of the present invention may comprise one or more
washing steps. By
washing, the unreacted reagents can be removed. For example, when the solid
phase comprises a strip of
microtiter wells, a washing substance or buffer is contacted with the wells
after each step. Examples of the
washing substance that can be used include 2-[N-moipholino]ethanesulfonate
buffer (IVIES), or phosphate
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buffered saline (PBS), etc. The pH of the buffer is preferably from about pH
6.0 to about pH 10Ø The buffer
may contain a detergent or surfactant, such as Tween 20.
The sandwich immunoassay can be carried out under typical conditions for
immunoassays. The typical
conditions for immunoassays comprise those conditions under which the pH is
about 6.0 to 10.0 and the
temperature is about 30 to 45 C. The pH can be regulated with a buffer, such
as phosphate buffered saline
(PBS), a triethanolamine hydrochloride buffer (IBA), a Tris-HC1 buffer or the
like. The buffer may contain
components used in usual immunoassays, such as a surfactant, a preservative
and serum proteins. The time of
contacting the respective components in each of the respective steps can be
suitably established depending on
the antigen to be measured, the antibody to be used, and the type of the solid
phase or the like.
Kits
The materials for use in the methods of the present invention are suited for
preparation of kits produced
in accordance with well known procedures. The invention thus provides kits
comprising agents, which may
include gene-specific or gene-selective probes and/or primers, for
quantitating the expression of the disclosed
genes for predicting prognostic outcome or response to treatment. Such kits
may optionally contain reagents for
the extraction of RNA from tumor samples, in particular fixed paraffin-
embedded tissue samples and/or
reagents for RNA amplification. In addition, the kits may optionally comprise
the reagent(s) with an identifying
description or label or instructions relating to their use in the methods of
the present invention. The kits may
comprise containers (including microtiter plates suitable for use in an
automated implementation of the method),
each with one or more of the various reagents (typically in concentrated form)
utilized in the methods, including,
for example, pre-fabricated microarrays, buffers, and the like.
The methods provided by the present invention may also be automated in whole
or in part. The
invention further provides kits for performing an immunoassay using the FAS
antibodies of the present
invention.
All aspects of the present invention may also be practiced such that a limited
number of additional
genes that are co-expressed with the disclosed genes (e.g., one or more genes
from the GPEPs or FAS), for
example as evidenced by high Pearson correlation coefficients, are included in
a prognostic or predictive tests in
addition to and/or in place of disclosed genes.
The invention is further illustrated by the following non-limiting examples.
EXAMPLES
Example 1. Design of the Investigation
The pre-clinical study was designed to show the diagnostic superiority of the
FAS assay by
predicting outcomes for patients with early stage (node negative) breast
carcinoma. In particular it
was designed to estimate the proportion of breast cancer cases correctly
diagnosed as lymph node
positive by FAS scores but earlier classified as lymph node negative by
morphologic methods. The
sensitivity of the assay was also determined by evaluating the proportion of
cases incorrectly
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diagnosed by FAS scores on cases correctly classified as lymph node negative
by morphologic
methods.
Cases with negative lymph node biopsies with known follow-up outcomes of non-
metastatic
(NM or MO; GOOD outcome) or metastatic (M1 or M2; POOR outcome) were diagnosed
by both
morphologic methods and FAS assay. Since the population chosen was well-known
with respect to
outcome, the proportion of M versus NM had to be considered a design
parameter.
All study cases, M and NM, were previously diagnosed by morphological methods.
Since
morphologic methods already missed the M cases it was assumed that the
probability that an (M) case
will be classified as non-morphologic on repeated morphologic diagnosis was
very small.
Example 2. Patient Population and Specimen Selection
The study consisted of 360 cases of formalin-fixed, paraffin embedded
infiltrating (invasive)
ductal carcinoma (IDC) of the breast originally obtained from patients during
the years 1980-1985.
All patients must have had lymph node biopsies or excision featuring six or
more removed lymph
nodes which showed no evidence of metastatic carcinoma (poor outcome).
Patients with papillary,
colloid, medullary and non-infiltrating (non-invasive) carcinoma were
excluded.
The population of patients represented two groups of 180 patients per
population. The first
population (A) consisted of a sub-population of the above patient group in
which all patients were
correctly classified via morphological parameters (e.g., each patient at the 5
year point is alive,
without disease, as verified by patient medical records, etc). These types of
patients were classified as
GOOD outcome patient population. The second (B) sub-population consisted of
patients that were
correctly classified at the time breast cancer was diagnosed but went on to
have a metastatic event
within five years of the original diagnosis. This totaled 360 patients in a
double-blind multi-center (3
sites, I, II and III) pre-clinical study.
Review of the surgical pathology slides and reports were performed in order to
determine and
record tumor sizes and grade. In addition to recording the largest tumor
diameter listed in the surgical
pathology report, measurement of the largest diameter of continuous tumor on
the microscope slide
was also provided. Tumors were graded according to the modified Scarff-Bloom-
Richardson system.
The modified Bloom-Richardson-Elston grading system is also called the
Nottingham system. In this
system the cells and tissue structure of the breast cancer are examined
histopathologically to
determine how aggressive the cancer is. The test comprises the observation of
three features when
determining a cancer's grade: (a) the frequency of cell mitosis (rate of cell
division), (b) tubule
formation (percentage of cancer composed of tubular structures), and (c)
nuclear pleomorphism
(change in cell size and uniformity). Each of these features is assigned a
score ranging from 1 to 3 (1
indicating slower cell growth and 3 indicating faster cell growth). The scores
of each of the cells'
features are then added together for a final sum that range between 3 and 9. A
tumor with a final sum
of 3, 4, or 5 is considered a Grade I tumor (well-differentiated). A sum of 6
or 7 is considered a Grade
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II tumor (moderately-differentiated), and a sum of 8 or 9 is a Grade III tumor
(poorly-differentiated).
Grades were reported as Grade I (scores 3-5); Grade II (scores 6-7) and Grade
III (scores 8-9).
Additional morphologic features which were prognostic recorded included
primary tumor
therapy type (lumpectomy verses mastectomy with or without radiation), status
of the resection
margin, evidence of skin, nipple or lymphatic vessel invasion, and
determination of the extent of an
in-situ component if present should were also included.
A representative 5 micron tissue section taken from a cellular area of
infiltrating carcinoma
was analyzed for loss of FAS expression or low nuclear expression of FAS. All
cases were recorded
as the percentage of cells that are positive or negative as a result of the
scoring method of FAS. All
results were reported on case report forms provided in the protocol. Each
sample had the following
tumor markers data available: Estrogen and Progesterone receptor, the Dako
HERcept Test m HER-2/neu analysis, cathepsin D, and in cases where available,
DNA ploidy analysis
(either by flow cytometry of image analysis).
All cases accrued to the study had documented clinical follow-up for a minimum
of five
years. For each case, follow-up at year five (5) was documented as: (a) alive,
well and free of disease;
(b) alive, recurrent disease; (c) dead from disease; (d) dead, from other
causes with no evidence of
recurrence. For patients with recurrent disease, the method of determining
recurrence (biopsy proven
versus other method) and method of determining death from disease (autopsy or
no autopsy) were
also documented.
Example 3. Materials
All supplies, the FAS detection kit of the invention and all primary and
secondary reagents
necessary to perform the assay on the 360 breast cancer cases were provided to
the investigators at
two sites. They were also provided with 10 paraffin sections with a thickness
of 4 microns of selected
tumor rich tissue blocks featuring invasive (infiltrating) breast carcinoma.
The investigators performed the morphologic prognostic analysis on the primary
tumors,
confirmed the lymph node negative status, and obtained and recorded the
estrogen and progesterone
receptor status. The investigators also confirmed the clinical outcome
analyses according to the
protocol for each patient.
Control slides that were reflective of all possible positive and negative
interpretations were
provided.
Proficiency evaluations were also performed and included twenty (20) samples
at three sites
(proficiency only) where the FAS status was already determined.
Example 4. Statistical Analyses and Outcomes
The results of the scoring of the FAS loss of expression was analyzed using
multivariate
analysis relative to clinical outcome and other prognostic marker analyses
determined for the other
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cases included in the pre-clinical study. Multivariate analysis was done using
both logistic regression
and Cox regression (proportional hazards) model.
The goal of this statistical analysis was to assess whether FAS can predict
outcome of patients
with early stage breast cancer based on cases that ALL have been classified as
GOOD by careful
morphological methods. Cases classified as POOR by morphological methods were
not included in
this study.
In the population of cases available for study, approximately 70% had indeed a
GOOD
outcome as determined by follow-up. But 30% of cases had POOR outcome and thus
were incorrectly
classified by morphological methods. From the total available cases, 360 cases
were randomly
selected with the restriction that 180 cases had POOR outcome in follow up and
180 cases had GOOD
outcome in follow up.
These data were intended to answer the following main questions: (1) Can FAS
correctly
classify POOR outcome cases that by morphological methods are missed; and (2)
Does FAS agree on
cases correctly classified as GOOD by morphological methods?
The analyses showed that FAS measurements scores correctly classified 72% of
the 30%
POOR outcomes that morphological methods missed (or 23% out of 30%).
Similarly, FAS
measurement scores correctly classified 90% of the 70% the morphological
methods correctly
classified as GOOD outcome (or 61% out of 70%).
The data analyzed in this report consisted of 180 cases with known Poor
Outcome and 180
cases with known Good Outcome. All 360 cases were of women who had originally
been classified as
Stage I by a panel of physicians. Cases classified as POOR by morphological
methods were not
available.
The AGE of the patient was available as a potential risk factor, but one that
was not at all
significant in contributing to an improved prediction. For each case the only
information available
other than the FAS-score was whether or not Poor Outcome was found after
original classification as
Stage I.
Cases were classified according to FAS expression according to the scoring
method shown in
Tables 2 and 3. A dual classification system was used for each specimen where
the nuclear stain
intensity is listed first, followed by the percent positivity of the cells
stained listed second.
Table 2: Classification according to Nuclear Stain
Intensity of Nuclear Stain SCORE
None 0
Light brown 1
Light-Medium brown 2
Medium-Dark brown 3
Dark brown 4
Table 3: Classification according to Percent Positivity
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Percent Positivity of SCORE
Nuclear Stain
None 0
<13% 1
13-35% 2
>35-75% 3
>75% 4
Example 5. Case classifications from multiple sites
The data for the first twenty cases are shown in Tables 4-6. Each case
material was tested at
three sites, Site I, Site II and Site III. For the present analysis the scores
from the three sites were first
separated into a separate staining intensity and percent positivity score. The
separate scores were
averaged over the three sites into a staining intensity and percent positivity
average score. A dual
classification system was used for each specimen where the nuclear stain
intensity is listed first,
followed by the percent positivity of the cells stained listed second.
Three cases in the Good Outcome group did not have values for one of the
sites. These were
averaged over two sites only. This accounts for an average score of 3.5,
whereas all others scores
change by thirds.
Table 4. Site I
SPECIMEN 1 2 3 4 5 6 7 8 9 10
Invasive Ductal Carcinoma
Epithelium 2/3 1/2 2/3 2/3 2/3 2/3 2/3 2/3 2/3 2/3
Ductal 2/3 1/2 2/2 2/2 1/2 2/3 1/2 1/2 2/3 1/2
Components
Inflammatory 2/3 0- 2/3 2/3 2/3 1/2 2/3 2/3 2/3 2/3
Cells user
error
Normal Breast
Epithelium 2/3 3/4 2/3 2/3 2/3 2/3 2/3 3/3 3/3 3/3
Ductal 3/3 3/3 3/3 3/3 3/3 2/3 3/4 3/4 2/3 3/4
Components
Table 5. Site II
SPECIMEN 11 12 13 14 15 16 17 18 19 20
Invasive Ductal Carcinoma
Epithelium 2/3 1/2 2/2 2/3 2/2 2/3 2/3 2/3 2/3 2/3
Ductal 2/2 1/2 2/3 2/2 1/2 2/3 1/2 1/2 2/3 1/2
Components
Inflammatory 2/3 1/1 3/3 2/3 2/3 1/2 2/3 2/3 2/3 2/3
Cells
Normal Breast
Epithelium 2/4 3/3 2/3 2/3 2/3 2/3 2/3 2/3 3/3 3/3
Ductal 3/3 3/4 3/3 2/3 3/3 3/3 3/4 3/4 2/3 4/4
Components
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Table 6. Site III
SPECIMEN 1 2 3 4 5 6 7 8 9 10
Invasive Ductal Carcinoma
Epithelium 2/3
2/2 2/3 2/3 1/2 2/3 2/3 1/2 2/3 2/3
Ductal 1/2
2/2 2/2 1/2 2/2 2/3 1/2 2/2 2/3 1/2
Components
Inflammatory 2/2 2/3 2/3 2/2 1/2 1/2 2/3 2/3 2/3 2/2
Cells
Normal Breast
Epithelium 3/3
3/3 2/3 2/3 2/3 4/4 2/3 2/3 3/4 3/4
Ductal 2/3
3/4 3/4 2/3 3/3 3/3 3/4 3/4 2/3 3/4
Components
Example 6. Discriminant Analysis (DA) and Logistic Regression (LR)
The statistical analysis involved in the present study included consideration
of several factors.
First the agreement between sites was examined. Simple correlations and kappa
scores of agreement
were used to examine the degree of agreement between sites. The analysis
showed that agreement was
not very satisfactory. Agreement between Positivity and Intensity within each
site was fairly high
(correlations in the .5 range), but agreement between sites was fairly low
(correlations in the .2 to .3
range). Sites I and III agreed more with each other than with Site II. As a
consequence, it was decided
to use average Intensity and Positivity scores across three sites. For three
cases data from one site was
missing. For those the average was calculated from the remaining two sites.
Data from at least two
sites were always available. Average Scores increased the probability of
correct classification
considerably.
Discriminant Analysis and Logistic Regression
Discriminant Analysis and Logistic Regression were both used to classify
cases. Both
Discriminant Analysis (DA) and Logistic Regression (LR) result in case scores
that are turned into
estimated probabilities of group membership. The two groups here are GOOD
outcome and POOR
outcome. The two methods yielded slightly different probabilities of group
membership, but virtually
the same classification of the 360 cases into POOR and GOOD. Other methods,
with different
assumptions, such as Cluster Analysis and Fuzzy Partitioning also yielded very
similar results.
For discriminant analysis based on randomly split data, the data were randomly
split into two
sets each about half the original data set. DA and LR parameters were
calculated based on one half of
the data and used to predict the classification of the other half of the data.
The agreement between
split and full data analysis was very close.
Probability calculations
Calculations to determine the probability that FAS measurement scores
correctly classified
POOR and GOOD outcomes were performed. The available data set only contained
cases that
morphological methods already identified as GOOD. Therefore resulting
probabilities are restricted to
such cases. In this step, which is combined with Step 2 on statistical
packages, the probabilities of
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group membership are used to predict (or classify) cases as GOOD or POOR
resulting in predicted
group membership for each case. Predicted group membership can be cross-
classified with actual
group membership in a 2X2 contingency table.
From the results, the following probabilities ("P") of misclassification were
estimated:
P[FAS predicts poor outcome l good outcome group] = 23/180 = .128
P[FAS predicts good outcome l poor outcome group] = 42/180 = .233
The backward conditional probabilities estimate the probability of an outcome
given a certain
FAS measurement score test result:
P[Good Outcome 1FAS good outcome] = 157/(157+42) = .788
P[Poor Outcome 1FAS poor outcome] = 138/(138+23) = .857
The sample contains exactly 180 cases with GOOD outcome and 180 cases with
POOR
outcome (50% GOOD, 50% POOR) while the actual population contained
approximately 70%
GOOD and 30% POOR outcomes. The population adjusted backward conditional
probabilities were:
P[Good Outcome 1FAS good outcome] = .61/.68 = .90
P[Poor Outcome 1FAS poor outcome] = .23/.32 = .72
These results show that FAS measurement score agrees with morphological
methods when
they are correct. FAS predicted GOOD outcome for 87% of the GOOD outcome
cases.
Morphological methods predicted GOOD for 100% of the cases, for these are the
only ones included
in the study. The results also show that FAS measurement score disagrees with
morphological
methods when they are wrong. Consequently, FAS measurement score correctly
classifies 76.7% of
all the POOR outcome cases that morphological methods falsely called GOOD.
Development of Scoring Cut-Offs Based on Classification Probabilities
Statistically, whenever the P[GOOD] >.5, then a case is classified as GOOD.
This criterion
yields cut-off points based on Logistic Regression as shown in Table 7. The
data are based on the
scoring system outlined in Tables 2-3 and the data of Tables 4-6 and includes
the dual scores for all of
the epithelial, ductal and inflammatory cells for cancer patients and the
epithelial and ductal
components for normal tissues.
Table 7: Boundary Pairs for (Intensity, Positivity) readings of FAS expression
Classify as Good outcome IF
Stain Intensity is at AND Positivity is at least
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least
2.45 3.69
2.69 3.23
3.10 3.00
3.23 2.55
3.69 2.45
3.98 2.00
DA and LR yield slight different cut-offs. DA is more willing to classify a
case as GOOD
than is LR.
Comparison With Her-2/neu and Estrogen Receptor Tests
The results from these two tests were cross-tabulated with actual group
membership and also
included as predictor terms in LR.
Neither of these tests reached the correct classification probabilities of the
average FAS-score,
nor were they helpful as additional terms in a LR model. HER-2/neu was not
significant and ER
actually counterproductive, reducing the correct classification probabilities
noticeably.
Discriminant Analysis of all Cases
Discriminant analysis is a multivariate statistical technique used to classify
subjects into two
or more non-overlapping populations. In traditional discriminant analysis one
uses the known group
membership of the subjects to derive a linear function of the score variables
to optimize prediction of
group membership. From the scores of the discriminant function one can derive
a probability of group
membership. Clustering and Logistic Regression are alternative methods that
also calculate
probabilities of group membership for each case. These differ from those
obtained in discriminant
analysis.
In the FAS study there were two populations, Poor (Outcome) and Good
(Outcome).
Accordingly, discriminant analysis calculates two probabilities.
1. P[Poor Outcome] is the estimated probability that a case belongs to the
Poor Outcome group.
2. P[Good Outcome] is the estimated probability that a case belongs to
the Good Outcome
group.
Hence, P[Poor Outcome] + P[Good Outcome] = 1.
If P[Poor Outcome] > P[Good Outcome], i.e., if P[Poor Outcome] > .5, then a
case is
predicted to belong to the Poor Outcome group and vice versa. A variable "Pred
group" is the
prediction of discriminant analysis as to group membership.
The formulas for all 360 cases are reminiscent of a quadratic regression
equation. Essentially
one calculates for each case a group score for the Good and the Poor group.
These formulas for the
group scores are given as Dist[good] and Dist[poor] respectively.
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Dist[good] = 60.7262835-14.779283*avgIntensity+4.97420821*avgIntensity^2 ¨
22.42291*avgPositivity+5.94409261avgPositivity^2 -
5.2174324*avgIntensity*avgPositivity
Dist [p o or] =37.8423625-10.183222*avgIntensity+4.97420821*avgIntensity^2 ¨
19.098415*avgPositivity+5.94409261avgPositivity^2 ¨
5.2174324*avgIntensity*avgPositivity
The scores are used to calculate the probability of group membership:
P[good] = exp {-.5*Dist[good]} /P [Sum]
P [poor] = exp {-.5*Dist[poor]}/P[Sum]
Where
P[Sum] = exp {-.5*Dist[good]} +exp {-.5*Dist[poor]}
Table 8 shows the results for subjects 1 and 2 of the Good Outcome group and
subjects 181
and 182 of the Poor Outcome group.
Table 8: Four Sample results of Discriminant Analysis
avg Prob Prob
avg (percent
case group (staining positivity) [Poor [Good Pred
group
intensity) Outcome] Outcome]
1 good 3.223 3.223 0.145 0.828 Good
Outcome
2 good 3.223 3.223 0.145 0.843 Good
Outcome
181 poor 3.223 2.669 0.242 0.669 Good
Outcome
182 poor 2.223 3.100 0.728 0.261 Poor Outcome
As can be seen, for cases 1, 2, and 182 the actual group membership variable
"group" agrees
with "Pred group" and so they are correctly classified as GOOD, GOOD and POOR
Outcomes
respectively. Case 181 shows a difference between "group" and "Pred group" and
represents a
misclassification.
A cross-classification of actual "group" membership versus "Pred group"
results Table 9. The
table shows that discriminant analysis would have classified 138 of the 180
Poor Outcome cases
correctly, but would have called 42 Good Outcome. This is a false negative
rate of 23.3%. Of the
Good Outcome cases, discriminant analysis would have predicted 157 correctly,
with 23 false
positives ¨ a 12.8% error rate. The overall error rate is 65 out of 360 or
18.1%.
Table 9: Cross-classification of predicted group membership with actual group
membership of
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180 GOOD and 180 POOR cases based on Discriminant Analysis
group
Pred Poor Good Total
Group Poor Outcome 138 23 161
Good Outcome 42 157 199
Total 180 180 360
The Kappa coefficient of intrarater reliability is 0.599639 with a Std Err of
0.040286, which is
fairly high and statistically significant.
From this Table one can estimate the following probabilities of
misclassification:
P[FAS predict poor outcome l good outcome group] = 23/180 = .128
P[FAS predict good outcome l poor outcome group] = 42/180 = .233
The complete estimated classification probabilities are in Table 10:
Table 10: Estimated prediction probabilities
group
Counts Poor Good Total
Poor Outcome .767 .128 .44
Good Outcome .233 .872 .55
a
o
;- Prob in Sample .5 .5 1
to
-o
eL) EST. Prob in Pop .3 .7 1
;-
a
The backward conditional probabilities estimate the probability of an outcome
given a certain
FAS test result:
P[Good Outcome 1FAS good outcome] = 157/(157+42) = .788
P[Poor Outcome 1FAS poor outcome] = 138/(138+23) = .857
The sample contains exactly 180 cases with GOOD outcome and 180 cases with
POOR
outcome (50% GOOD, 50% POOR. These probabilities are based on the assumption
that the sample
proportion of GOOD and POOR outcomes reflects the population proportions.
The sample was taken from a population that morphological methods classified
entirely as
GOOD. This population contained approximately 30% poor outcomes. So to
estimate the success of
FAS classification one must adjust the classification probabilities from the
sample probabilities to
those in the population (70% GOOD, 30% Poor) by multiplying the estimated
probabilities
accordingly. For example, .767*.3 = .23 = P[FAS classifies a cases as poor and
that case has indeed
poor outcome in the population]. This yields the following adjusted table of
probabilities:
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Table 11: Estimated prediction probabilities adjusted for 70/30 representation
of
GOOD/POOR in available samples
group
Counts Poor Good Total
Poor Outcome .23 .09 .32
a
o Good Outcome .070 .61 .68
;-
to
-o
eL) EST. Prob in Pop .30 .70 1
;-
a
The adjusted backward conditional probabilities are:
P[Good Outcome 1FAS good outcome] = .61/.68 = .90
P[Poor Outcome 1FAS poor outcome] = .23/.32 = .72
These probabilities state that in a population containing only cases that had
been identified as
GOOD by morphological methods, GOOD results on a FAS test carries with it a
0.90 probability that
the case is indeed GOOD. Likewise, a POOR result on a FAS test carries with it
a 0.72 probability
that the case is indeed POOR.
Using the discrimination formulas the following probabilities of group
membership can be
arrived at assuming average scores. The predicted Good Outcome Group consists
of scores (Intensity,
Positivity) = {(3,3), (3,4), (4,2), (4,3), (4,4)}. These four groups have a
P[good] > 0.5.
Logistic Regression
Logistic Regression yields virtually the same classification as did
discriminant analysis. Only
one case, with site scores of (4/3, 2/1, 4/3) and average scores of (3.33,
2.33) would have been
classified as Good with discriminant analysis (P[poor] =.475), and as Poor
with logistic regression
(P[poor]=.509.
Example 7. Analysis of Other Variables
Age as Additional Covariate
As additional variable the age of each case (patient) was entered into the
Logistic Regression
model. It was determined that age did not produce a significant contribution
(p=.39).
HER-2/Neu
A cross-classification of actual "group" membership versus HER results was
performed. The
result showed that HER-2/Neu would have classified 85 of the 180 Good Outcome
cases as negative,
and would have called 95 positive. This is a false positive rate of 52.8%
(compared to 23.3% for
average FAS). Of the Poor Outcome cases, HER-2/Neu would have predicted 109
(60.5%) correctly,
with 71 false negatives ¨ a 39.4% error rate compared to 12.3% for the FAS
average. The overall
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error rate is (95+71) out of 360 or 46.1%. This simple classification shows
that HER-2/Neu is
considerably inferior to the FAS measurement scores in prediction outcome. In
fact, there is hardly a
noticeable reduction in error, although the results are significant.
ER-Estrogen Receptor
A cross-classification of actual "group" membership versus ER was performed.
The data
showed that ER would have classified 90 of the 180 Good Outcome cases as
negative, and would
have called 90 positive. This is a false positive rate of 50% (compared to
23.3% for average FAS). Of
the Poor Outcome cases, ER would have predicted 128 (71.1%) correctly, with 52
false negatives ¨ a
28.9% error rate compared to 12.3% for the FAS average. The overall error rate
was (90+52) out of
360 or 39.4% compared with 18.1% of the FAS average. This classification
showed that ER is better
than HER-2Neu in classifying outcome correctly, but still considerably
inferior to the FAS average
scores. In fact, there is small reduction in error, and the results are
significant. Practically they appear
of very limited value.
In summary, FAS appears to have considerable diagnostic value above and beyond
morphological methods. FAS measurement found over 85% of the POOR cases
morphological
methods missed and was in agreement 90% when morphological methods correctly
classified as good.
HER-2/neu and ER proved to inferior to FAS on this data set. The age of
patient was not a significant
predictor. Statistical methods based on a variety of assumptions seem to
produce similar results.
Example 8. FAS Standard ELISA Colorimetric or Chemiluminescence Protocol
The following is an outline of the procedures for the development,
optimization and
documentation of novel antibodies using serial dilution and automated
Immunohistochemistry (IHC)
staining.
Definition
PBS: Phosphate Buffered Saline is a buffer solution commonly used in
biological research. It
is a water-based salt solution containing sodium chloride, sodium phosphate,
and (in some
formulations) potassium chloride and potassium phosphate. The buffer helps to
maintain a constant
pH. The osmolarity and ion concentrations of the solution usually match those
of the human body
(isotonic).
Equipment
Ventana Benchmark XT Staining System or equivalent
Materials
PPE: personal protective equipment
Microcentrifugr tubes
15 mL conical tubes
PBS or diluent
Pipette
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Pipette tips
FAS Antibodies
Procedure
1. Coat wells on a 96 well microplate with 100 [LI/well of coating
antibody diluted in PBS.
2. Incubate plate overnight at 4 C, covered with plate sealer.
3. Wash plate 5 times with 300 ul of PBS-T (0.05% Tween 20) on Wellwash Versa
Plate
washer (Thermo).
4. Block plates with 300 [LI/well of ELISA Blocker Blocking Solution
(Thermo) for 2 hours
at 23 C with shaking at 100 rpm in Incubating Microplate Shaker (VWR) covered
with
plate sealer.
5. Wash plates 5 times with PBS-T 300 [LI/well on plate washer.
After each washing step, tap plate onto kimwipes on bench to remove any excess
liquid.
6. Load 100 [L1 of standards or samples freshly diluted in PBS-T 23 C on
plate shaker with
100 rpm agitation for 2 hours, covered with plate sealer.
Prepare protein standards ahead of time on ice.
7. Freshly prepare a 7-point dilution. e.g. from 400 ng/ml to 1 ng/ml in 1%
BSA/PBS-T.
8. Wash plate 5 times with PBS-T 300u1/well on plate washer.
9. Incubate 100 [LI/well of biotinylated detection antibody diluted in PBS to
appropriate
concentration for 2 hours at 23 C on plate shaker with 100 rpm agitation,
covered with
plate sealer.
10. Wash plate 5 times with PBS-T 300u1/well on plate washer.
11. Incubate 100 [LI/well of streptavidin-HRP (R&D Inc.), 1:200 dilution in
PBS at 23 C on
plate shaker with 100 rpm agitation for 20 minutes, covered with plate sealer.
12. Wash plate 5 times with PBS-T 300u1/well on plate washer.
For colorimetric measurement, add 100 ul of Tetrarnethylbenzidine substrate
solution
(Thermo) to each well.
13. Incubate for 20 minutes at room temperature in the dark.
14. Add 50 ul of acidic Stop solution (Thermo) to stop color development.
15. Determine the optical density of samples with BioTek FL800x plate reader
at 450 rim,
For chemiluminescence measurement, amplify signal by adding 100 id/well Gloset
Substrate (R&D Inc.), for 5 to 15 minutes at room temperature inside a BioTek
FL800x plate reader. Prepare Substrate ahead of time.Freshly prepare by mixing
Reagent A: Reagent B 1:2.
16. Set signal measured on BioTek FL800x fluorometer at 0.5 second read time
with
sensitivity, auto adjusted to highest point on standard curve, and set to a
reading of
400,000.
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It should be noted that ELISA Sandwich assays useful in the present invention
include those as described in PCT Publication PCT/U52010/046773 published
March 17, 2011, the
contents of which are incorporated here by reference in its entirety.
Example 9. Use of Commercial FAS Antibodies
IHC and ELISA assays may be performed using a commercial anti-FAS antibody.
For IHC
the antibodies used are human anti-FASN Antibody, Affinity Purified (Catalog
No. A301-324A)
from Bethyl Laboratories (Montgomery, TX). For ELISA studies, the antibodies
used are the Fatty
Acid Synthase Antibody Pair (Catalog No. H00002194-AP11) from Novus
Biologicals (Littleton,
CO). The pair contains a Capture antibody which is rabbit affinity purified
polyclonal anti-FASN (100
ug) and a Detection antibody which is mouse monoclonal anti-FASN, IgG1 Kappa
(20 ug).
Example 10: Preparation of Anti-FAS Monoclonal Antibodies
Anti-FAS antibodies and an immunohistochemical ELISA assay employing the
antibodies are
disclosed in PCT Publication PCT/U52010/030545 published October 14, 2010, and
PCT/US2010/046773 published March 17, 2011, respectively. The contents of each
are incorporated
here by reference in their entirety.
Briefly, four murine monoclonal antibodies were prepared by immunizing SCID
mice with
synthetic FAS peptides, and establishing hybridomas according to the general
procedure described by
Iyer et al., Ind. J. Med. Res., 123:651-564 (2006). Each mouse was immunized
with one peptide of
SEQ ID NOs 1-5.
FAS Peptides:
SEQ ID NO. 1 VAQGQWEPSGXAP
SEQ ID NO. 2 PSGPAPTNXGALE
SEQ ID NO. 3 TLEQQHXVAQGQW
SEQ ID NO. 4 EVDPGSAELQKVLQGD
SEQ ID NO. 5 ELSSKADEASELAC
Humanized monoclonal antibodies were prepared as described by Carter et al.,
Proc. Nad
Acad. Sci. USA, 89:4285-89 (1992) from monoclonal antibodies derived from
hybridomas A,B, D and
E. The humanized monoclonal antibodies (MAbs) are referred to hereinafter as
FAS 1, FAS 2, FAS 4
(ATCC Deposit No: PTA-10811) and FAS 5(ATCC Deposit No: __ ), respectively.
Example 11: FAS Detection in Nipple Aspirate Fluid
Ductal fluid was collected by nipple aspiration using a modified breast pump
used to express
milk from women with breast cancer. The modified breast pump consisted of a
plastic cup connected
to a section of polymer tubing which was then attached to a standard syringe
in order to create a
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gentle vacuum. Before aspiration, the nipple was cleansed in order to remove
keratin plugs, and then
with an alcohol pad. The breast was allowed to dry before a warm moist cloth
was placed on the
breast for about 1-2 minutes. After the damp cloth was removed from the
breast, the breast was
gently massaged from the chest wall toward the nipple from about a minute. The
suction cup was
then placed over the nipple and the plunger of the syringe was withdrawn to
the 5- to 10-ml level until
ductal fluid was visualized. The fluid droplets were collected and sample
volumes of the nipple
aspirate fluid were recorded.
After collection, the nipple aspirate fluid samples were rinsed into
centrifuge tubes containing
500 ILEL of sterile PBS supplemented with protease inhibitors 442-aminoethy1]-
benzenesulfonylfluoride-HC1 (0.2 mmol/L), leupeptin (50 g/mL), aprotinin (2
g/mL), and DTT (0.5
mmol/L). The samples were then centrifuged at 1500 rpm for 10 minutes in order
to remove insoluble
materials. The supernatant was collected in 50 L aliquots which were processed
to determine the
intensity of the nuclear stain. The intensity level for each sample was
classified using the
classification system listed in Table 2 in order to determine the expression
level of FAS contained in
the samples.
35 nipple aspirate samples were collected and analyzed, as described above, in
order to
determine the levels of FAS expression in women with breast cancer. The
samples collected from
women over 50 years of age who had infiltrating ductal carcinoma were found to
have a decreased
chance of survival after 5 years when the expression of FAS in their nipple
aspirate fluid was greater
than 1. Table 12 is a listing of the 35 nipple aspirate samples collected and
analyzed.
Table 12: FAS Expression Levels from Nipple Aspirate Fluid Samples
Chemo- Radiation 5 year
Age Histology Stage FAS Surgery therapy Therapy Survival
Lobular
39 carcinoma 2 1 Mastectomy CMF Radiation Yes
Lobular
39 carcinoma 2 2 Mastectomy CMF Radiation Yes
Matched
benign
39 specimen 2 Mastectomy Radiation Yes
Infiltrating
ductal
40 carcinoma 2 2 Biopsy Radiation Yes
Tubular
41 carcinoma 2 1 Biopsy Radiation Yes
Tubular
41 carcinoma 2 1 Biopsy Radiation Yes
Matched
benign
41 specimen 1 Biopsy Radiation Yes
Infiltrating
ductal
46 carcinoma 3 3 Mastectomy 5-FU Radiation Yes
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Infiltrating
ductal
46 carcinoma 3 3 Mastectomy 5-FU Radiation Yes
Matched
benign
46 specimen 3 Mastectomy Radiation Yes
Infiltrating
ductal
52 carcinoma 1 2 Biopsy Radiation No
Infiltrating
ductal
52 carcinoma 1 2 Biopsy Radiation No
Matched
benign
52 specimen 2 Biopsy Radiation Yes
Infiltrating
ductal
55 carcinoma 1 1 Biopsy Radiation Yes
Matched
benign
55 specimen 1 Biopsy Radiation Yes
Infiltrating
ductal
61 carcinoma 2 1 Mastectomy
Adria/CMF Radiation Yes
Infiltrating
ductal
61 carcinoma 2 1 Mastectomy
Adria/CMF Radiation Yes
Scirrhous
61 carcinoma 1 1 Biopsy Radiation Yes
Lobular
65 carcinoma 2 2 Biopsy Radiation Yes
Lobular
65 carcinoma 2 2 Biopsy Radiation Yes
Lobular
65 carcinoma 2 2 Biopsy Radiation Yes
Medullary
65 carcinoma 2 3 Mastectomy CMF Radiation Yes
Medullary
65 carcinoma 2 3 Mastectomy CMF Radiation Yes
Infiltrating
ductal
69 carcinoma 3 4 Mastectomy
Adria/CMF Radiation No
Infiltrating
ductal
69 carcinoma 3 4 Mastectomy
Adria/CMF Radiation No
Infiltrating
ductal
86 carcinoma 3 4 Biopsy Radiation Yes
Infiltrating
ductal
86 carcinoma 3 4 Biopsy Radiation Yes
Infiltrating
ductal
Unk carcinoma 2 2 Mastectomy CMF Radiation No
Infiltrating
ductal
Unk carcinoma 2 2 Mastectomy CMF Radiation No
Infiltrating
Unk ductal 2 1 Mastectomy CMF Radiation Yes
41
CA 02852757 2014-04-16
WO 2013/059105
PCT/US2012/060187
carcinoma
Infiltrating
ductal
Unk carcinoma 2 1 Mastectomy CMF Radiation Yes
Infiltrating
ductal
Unk carcinoma 1 1 Mastectomy Radiation Yes
Infiltrating
ductal
Unk carcinoma 1 1 Mastectomy Radiation Yes
Scintious
Unk carcinoma 1 1 Mastectomy Radiation Yes
Scintious
Unk carcinoma 1 1 Mastectomy Radiation Yes
For women diagnosed with infiltrating ductal carcinoma the data show that
irrespective of
cancer stage, type of surgery or diameter of tissue sample, where FAS levels
in nipple aspirates are 2
or greater and the resected margin is positive, there is a less than 5 year
survival rate. The data are
shown in Table 13.
Table 13: Five year survival when FAS greater than 2 and Positive Resected
Margin
Resected Radiation 5 year
Age Diameter Surgery Margin Stage FAS Therapy Chemotherapy Survival
52 2.0cm Biopsy Positive 1 2 Radiation No
52 2.0cm Biopsy Positive 1 2 Radiation No
Unk Mastectomy Positive 2 2 Radiation CMF No
Unk Mastectomy Positive 2 2 Radiation CMF No
69 6.0cm Mastectomy Positive 3 4 Radiation Adria/CMF No
69 6.0cm Mastectomy Positive 3 4 Radiation Adria/CMF No
42
